Multiphase systems and uses thereof

ABSTRACT

A multi-phase system includes a phase-separated solution comprising at least two phases, each phase having a phase component selected from the group consisting of a polymer, a surfactant and combinations thereof, wherein at least one phase comprises a polymer, wherein the phases, taken together, represent a density gradient. Novel two-phase, three-phase, four-phase, five-phase, or six-phase systems are disclosed. Using the disclosed multi-phase polymer systems, particles, or other analyte of interest can be separated based on their different densities or affinities.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent Ser. No.13/817,976, filed Aug. 14, 2013, which is a National Stage Entry ofPCT/US2011/48673, filed Aug. 22, 2011, which claims priority to U.S.Provisional Patent Application No. 61/375,532, filed on Aug. 20, 2010,the contents of all of which are hereby incorporated by reference in itsentirety. This application is also related to the followingapplications, filed concurrently herewith, the entire contents of whichare incorporated herein by reference:

PCT Patent Application No. PCT/US2011/048678, filed on Aug. 22, 2011,entitled “DENSITY-BASED SEPARATION OF BIOLOGICAL ANALYTES USINGMULTIPHASE SYSTEMS;”

PCT Patent Application No. PCT/US2011/048675, filed on Aug. 22, 2011,entitled “MULTIPHASE SYSTEMS HAVING MULTIPLE PHASE PROPERTIES;” and

PCT Patent Application No. PCT/US2011/048676, filed on Aug. 22, 2011,entitled “MULTIPHASE SYSTEMS FOR ANALYSIS OF SOLID MATERIALS.”

INCORPORATION BY REFERENCE

All non-patent literature, patents, patent applications and publicationscited herein are hereby incorporated by reference in their entirety inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the invention described herein.

BACKGROUND

It is known that the aqueous mixtures of two polymers such aspoly(ethylene glycol) (PEG) and dextran can separate spontaneously intotwo aqueous phases, called aqueous two-phase systems. Phase separationin aqueous solutions of polymers is an extraordinary and underexploredphenomenon. When two aqueous solutions of polymers are mixed, theresulting system is not homogeneous; rather, two discrete phases, orlayers, form. These layers are ordered according to density and arisefrom the mutual immiscibility of the polymers for one another. In thesesystems, each phase predominantly consists of water (upwards of 70-90%(w/v)), while the polymer component is present in concentrations rangingfrom micromolar to millimolar. A low interfacial tension and rapid masstransfer of water-soluble molecules across the boundary characterize theinterface between layers.

Previous studies of partitioning between aqueous phases have beenlimited to biphasic systems of immiscible polymers or inorganic saltsand have focused on applications in protein chemistry, cellpartitioning, and manufacturing. These Aqueous Two-Phase Systems(“ATPS”) are exemplified by the poly(ethylene glycol)-dextran,dextran-Ficoll systems, and a poly(ethylene glycol) system comprising(NH₄)₂SO₄.

Although numerous biphasic systems have been reported, there have beenrelatively few reports of polymer systems that exhibit multiphase, e.g.,more than two phases, separation: (i) poly(ethyleneglycol)-dextran-Ficoll, (ii) Triton X100-poly(ethylene glycol)-dextran,and (iii) poly(propylene glycol)-poly(ethylene glycol)-dextran-Ficoll.The four-phase system, however, is not entirely an aqueous systembecause the liquid poly(propylene glycol) that was employed in the assayis insoluble in water. Thus, while polymeric, this “four” phase systemcan be compared to the incorporation of an organic solvent orperfluorinated alkane into a three-phase system.

SUMMARY

Described herein are methods of separating or analyzing analytes ofinterest using multi-phase systems (“MPS”) comprising two or more phaseshaving different densities. In some embodiments, MPS as described hereinare used to separate analytes from each other or from impurities andother objects in the sample when the analytes migrate to phasescharacteristic of their densities, and in so doing, contact each phaseof the multi-phase system sequentially. As used herein, “sequentialcontact” means that the analyte contacts and interacts with only onephase (and its major phase component) at a time except at the interfacewhere the analyte may contact and interact with two adjacent phasessimultaneously. That is, the interaction of the analyte with the MPSoccurs when the MPS has already phase separated and not during theprocess of phase separation. In some embodiments, a multi-phase systemcomprising a phase component is used and the analyte contacts each phaseof the multi-phase system sequentially.

As used herein, a sample comprising one or more analyte is a mixture ofspecies that can be characterized, and differentiated, by a set ofphysical properties (e.g., density, size, or potentially others). Amulti-phase system can be designed to separate the analyte mixture intomultiple populations at the interfaces between phases. These populationscan be pure (a single species; ideal) or mixed. If mixed, thecompositions of the species captured at each interface share at leastone feature. For example, their densities are greater than that of thephase above but lesser than that of the phase below.

The multi-phase systems used in the methods disclosed herein comprisetwo or more phases that are phase-separated from each other, wherein ofthe two or more phases comprises a phase component. The phase componentis one or more selected from the group consisting of polymer,surfactant, or combinations thereof, wherein at least one of the phasecomponents is a polymer. The phases in the multi-phase system can beaqueous or organic. In some embodiments, at least one phase of themulti-phase system is aqueous and at least one phase of the multi-phasesystem is organic.

As used herein, MPS refers to a multi-phase system. When two or moresolutions containing a phase component are mixed, the resulting systemis not homogeneous; rather, two or more discrete phases, or layers,form. These layers are ordered according to density and arise from theexhibit limited interaction of the phase components with one another.The two or more phases or solutions, which exhibit limited interactionand form distinct phase boundaries between adjacent phases. Each phasecan be aqueous or non-aqueous. The non-aqueous phase comprises anorganic liquid or an organic solvent.

As used herein, AMPS refers to an aqueous multi-phase polymer system.ATPS refers to an aqueous two-phase polymer system.

As used herein, an aqueous multi-phase polymer system comprises two ormore polymer aqueous solutions or phases, which are phase-separated andin which at least two aqueous solutions each comprises a polymer. Insome embodiments, the aqueous multi-phase polymer system can be combinedwith one or more immiscible organic phases to form a multi-phase system.

As used herein, the polymer includes, but is not limited to, thehomopolymer, copolymer, terpolymer, and block copolymer, randomcopolymers and terpolymers of that polymer. As used herein, copolymerrefers to a polymer derived from two monomeric species; similarly, aterpolymer refers to a polymer derived from three monomeric species.Block copolymers include, but are not limited to, block, graft,dendrimer, and star polymers. The polymer also includes variousmorphologies, including, but not limited to, linear polymer, branchedpolymer, crosslinked polymer, and dendrimer systems. As an example,polyacrylamide polymer refers to any polymer including polyacrylamide,e.g., a homopolymer, copolymer, terpolymer, block copolymer orterpolymer of polyacrylamide. Polyacrylamide can be a linear polymer,branched polymer, crosslinked polymer, or a dendrimer of polyacrylamide.

In one aspect, a multi-phase system is described, comprising:

two or more phase-separated phases including at least a first and secondphases, wherein

-   -   each of the first and second phases comprises a phase component        selected from the group consisting of a polymer, a surfactant        and combinations thereof, wherein at least one of the first and        second phases comprises a polymer;    -   each said phase has an upper and a lower phase boundary; and    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and

an analyte contained within the phase-separated solution, wherein theanalyte is located predominantly at one of the phase boundaries.

The phase component is selected from the group consisting of a polymer,a surfactant and combinations thereof. The phase “combination” refers tothe combination of a polymer and a surfactant, a combination of two ormore polymers, a combination of two or more surfactants, or acombination of any number of polymers and any number of surfactants.

In any of the preceding embodiments, greater than 80% of the analyte islocated at one or more of the phase boundaries.

In any of the preceding embodiments, greater than 90% of the analyte islocated at one or more of the phase boundaries.

In any of the preceding embodiments, the system comprises more than oneanalyte and the additional analytes are located at the same or differentphase boundary.

In another aspect, a multi-phase system is described, comprising:

at least two phase-separated non-aqueous phases, wherein

-   -   each phase comprises a phase component selected from the group        consisting of a polymer, a surfactant and combinations thereof,    -   wherein at least one phase comprises a polymer; and    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient.

In any of the preceding embodiments, the multi-phase system comprisesthree or more phases.

In any of the preceding embodiments, the multi-phase system comprisesmore than one solvent, and wherein the phases comprising a commonsolvent share at least one boundary.

In any of the preceding embodiments, the phases comprises a commonsolvent and the common solvent is an aqueous solvent.

In any of the preceding embodiments, wherein the phases comprises acommon solvent which is an organic solvent.

In any of the preceding embodiments, the organic solvent is selectedfrom the groups consisting of liquid polymer, non-polar organic solvent,polar aprotic or protic solvent, supercritical fluid, fuel, oils, andfluorinated solvents, and combinations thereof.

In any of the preceding embodiments, the common solvent comprisesdichloromethane.

In any of the preceding embodiments, the multi-phase system comprisesphase separated solutions selected from the group consisting of organicsolutions, silicon oils, ionic liquids, fluorinated solvents, and liquidmetals.

In any of the preceding embodiments, the multi-phase system comprisesthree or more phases, and additional phase separated phases selectedfrom the group consisting of organic solutions, silicon oils, ionicliquids, fluorinated solvents, and liquid metals.

In any of the preceding embodiments, the aqueous phase is selected fromthe group consisting of water, isotopes of water, buffered water, seawater, irrigation water, mine effluent, colloidal solutions, emulsions,and combinations thereof.

In any of the preceding embodiments, the multi-phase system furthercomprises one or more additional analytes located predominantly at oneor more of the phase boundaries.

In any of the preceding embodiments, the analyte is insoluble in themulti-phase system.

In any of the preceding embodiments, the analyte has at least onedimension greater than 200 nm.

In any of the preceding embodiments, the analyte is selected from thegroup consisting of solid particles, an aggregate of particles, a liquidor gel immiscible in the solvent, a liquid crystal, crystallinematerials.

In any of the preceding embodiments, the analyte is selected from thegroup consisting of gem, bead, metal, glass, rock, mineral, crystal,plastic, bone, rubber, paper, fabric, coal, gases, polymer particles,and a combination thereof.

In any of the preceding embodiments, the polymer is selected from thegroup of homopolymers, random copolymers, block copolymers, graftcopolymers, ter-polymers, dendrimers, star polymers and combinationsthereof.

In any of the preceding embodiments, the polymer is linear, branchedand/or cross-linked.

In any of the preceding embodiments, the polymer is selected from thegroup consisting of dextran, dextran sulfate, chondroitin sulfate A,polysucrose, diethylaminoethyl-dextran, poly(2-vinylpyridine-N-oxide),poly(vinyl alcohol), poly(2-ethyl-2-oxazoline), poly(methacrylic acid),poly(ethylene glycol), polyacrylamide, polyethyleneimine, hydroxyethylcellulose, polyvinylpyrrolidone, carboxy-polyacrylamide, poly(acrylicacid), poly(2-acrylamido-2-methyl-1-propanesulfonic acid),poly(diallyldimethyl ammonium chloride), poly(styrene sulfonic acid),polyallylamine, alginic acid, poly(bisphenol A carbonate),polydimethylsiloxane, polystyrene, poly(4-vinylpyridine),polycaprolactone, polysulfone, poly(methyl methacrylate-co-methacrylicacid), poly(methyl methacrylate), poly(tetrahydrofuran), poly(propyleneglycol), and poly(vinyl acetate) and copolymers or terpolymer thereof.

In any of the preceding embodiments, the surfactant is selected from thegroup consisting of polysorbate,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate,polyoxyethylene-polyoxypropylene, 1-O-Octyl-β-D-glucopyranoside,4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,2-(Perfluoroalkyl)ethyl methacrylate, nonylphenol polyoxyethylene,N,N-dimethyldodecylamine N-oxide, polyethylene glycol dodecyl ether,sodium dodecyl sulfate, sodium cholate, benzylalkonium chloride anddodecyltrimethylammonium chloride.

In any of the preceding embodiments, one or more phases further comprisean additive selected from the group consisting of a salt, a misciblesurfactant, a co-solvent, an acid, a base, a miscible polymer, vitamin,drug, antibiotic, small molecule, dye, colloid, and flurophore.

In yet another aspect, an aqueous two-phase system is described,comprising: a phase-separated solution comprising two aqueous phases,wherein each of the phases comprises a phase component and has adifferent density and the phases, taken together, represent a densitygradient, and wherein the phase component combination of the two phasesis selected from the group consisting of:

Number Phase component combinations 1 poly(2-ethyl-2-oxazoline)poly(methacrylic acid) 2 poly(2-ethyl-2-oxazoline) poly(vinyl alcohol) 3poly(ethylene glycol) poly(methacrylic acid) 6 poly(ethylene glycol)poly(2-ethyl-2-oxazoline) 8 dextran poly(2-ethyl-2-oxazoline) 10 Ficollpoly(methacrylic acid) 11 Ficoll poly(vinyl alcohol) 12 Ficollpoly(2-ethyl-2-oxazoline) 15 polyacrylamide poly(methacrylic acid) 16polyacrylamide poly(acrylic acid) 18 polyacrylamidepoly(2-ethyl-2-oxazoline) 19 polyacrylamide poly(ethylene glycol) 20poly(diallyldimethyl ammonium poly(methacrylic acid) chloride 21poly(diallyldimethyl ammonium poly(acrylic acid) chloride 22poly(diallyldimethyl ammonium poly(vinyl alcohol) chloride 23poly(diallyldimethyl ammonium poly(2-ethyl-2-oxazoline) chloride 24poly(diallyldimethyl ammonium poly(ethylene glycol) chloride 25 dextransulfate poly(vinyl alcohol) 26 dextran sulfate poly(2-ethyl-2-oxazoline)28 chondroitin sulfate A poly(methacrylic acid) 29 chondroitin sulfate Apoly(vinyl alcohol) 30 chondroitin sulfate A poly(2-ethyl-2-oxazoline)31 polyethyleneimine poly(methacrylic acid) 32 polyethyleneiminepoly(2-ethyl-2-oxazoline) 33 polyethyleneimine poly(ethylene glycol) 34polyethyleneimine Ficoll 35 polyethyleneimine polyacrylamide 36polyvinylpyrrolidone poly(methacrylic acid) 39 poly(propylene glycol)poly(methacrylic acid) 41 poly(propylene glycol) polyacrylamide 42poly(2-acrylamido-2-methyl- dextran 1-propanesulfonic acid) 43poly(2-acrylamido-2-methyl- polyvinylpyrrolidone 1-propanesulfonic acid)44 poly(styrene sulfonic acid) poly(2-ethyl-2-oxazoline) 45 poly(styrenesulfonic acid) dextran sulfate 46 diethylaminoethyl-dextran poly(acrylicacid) 47 polyallylamine dextran sulfate 48 alginic acid poly(acrylicacid) 49 alginic acid poly(propylene glycol) 50 (hydroxypropyl)methylcellulose poly(diallyldimethyl ammonium chloride 51(hydroxypropyl)methyl cellulose poly(propylene glycol) 52carboxy-polyacrylamide poly(methacrylic acid) 53 carboxy-polyacrylamidepoly(vinyl alcohol) 54 carboxy-polyacrylamide polyethyleneimine 55hydroxyethyl cellulose dextran 56 hydroxyethyl cellulose Ficoll 57methyl cellulose Ficoll 58 Zonyl poly(methacrylic acid) 59 Zonyl dextran60 Zonyl polyacrylamide 61 Brij poly(2-ethyl-2-oxazoline) 62 Brij Ficoll63 Brij polyallylamine 64 Tween poly(methacrylic acid) 65 Tweenpoly(vinyl alcohol) 66 Tween poly(2-ethyl-2-oxazoline) 69 Tween Ficoll70 Tween polyacrylamide 71 Tween polyallylamine 72 Tween hydroxyethylcellulose 73 Triton poly(methacrylic acid) 74 Triton poly(acrylic acid)75 Triton poly(2-ethyl-2-oxazoline) 77 Triton Ficoll 78 Tritonpolyacrylamide 79 Triton polyallylamine 81 nonylphenol polyoxyethylenepoly(methacrylic acid) 82 nonylphenol polyoxyethylene dextran 831-O-Octyl-B-D-glucopyranoside poly(methacrylic acid) 841-O-Octyl-B-D-glucopyranoside poly(2-ethyl-2-oxazoline) 861-O-Octyl-B-D-glucopyranoside polyethyleneimine 87 Pluronicpoly(methacrylic acid) 88 Pluronic poly(vinyl alcohol) 89 Pluronipoly(2-ethyl-2-oxazoline) 90 Pluronic dextran 91 Pluronic Ficoll 92Pluronic polyacrylamide 93 Pluronic polyethyleneimine 94 sodium dodecylsulfate poly(acrylic acid) 95 sodium cholate poly(methacrylic acid) 96sodium cholate dextran sulfate 97 N,N-dimethyldodecylaminepoly(methacrylic acid) N-oxide 98 N,N-dimethyldodecylaminepolyacrylamide N-oxide 99 CHAPS poly(methacrylic acid) 100 CHAPSpoly(2-ethyl-2-oxazoline) 101 CHAPS poly(ethylene glycol) 102 CHAPSdextran 103 CHAPS Ficoll 104 CHAPS polyacrylamide 105 CHAPSpolyethyleneimine 106 CHAPS Pluronic 107 PVPNO PA 108 PVPNO PMAA 111PVPNO PEOZ 112 PVPNO PEG 116 PVPNO PEI 117 PVPNO Tween

In yet another aspect, an aqueous three-phase system is described,comprising:

a phase-separated solution comprising three aqueous phases, wherein eachof the phases comprises a phase component and has a different densityand the phases, taken together, represent a density gradient, andwherein the phase component combination of the three phases is selectedfrom the group consisting of:

Number Phase component combinations 1 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) 2 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) Ficoll 3 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) polyacrylamide 4 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(diallyldimethyl ammonium chloride 5poly(methacrylic acid) poly(2-ethyl-2-oxazoline) chondroitin sulfate A 6poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyethyleneimine 7poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Tween 8poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Triton 9poly(methacrylic acid) poly(2-ethyl-2-oxazoline)1-O-Octyl-B-D-glucopyranoside 10 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) Pluronic 11 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) CHAPS 12 poly(methacrylic acid) poly(ethyleneglycol) Ficoll 13 poly(methacrylic acid) poly(ethylene glycol)polyacrylamide 14 poly(methacrylic acid) poly(ethylene glycol)poly(diallyldimethyl ammonium chloride 15 poly(methacrylic acid)poly(ethylene glycol) polyethyleneimine 16 poly(methacrylic acid)poly(ethylene glycol) polyvinylpyrrolidone 17 poly(methacrylic acid)poly(ethylene glycol) Tween 20 18 poly(methacrylic acid) poly(ethyleneglycol) 1-O-Octyl-B-D-glucopyranoside 19 poly(methacrylic acid)poly(ethylene glycol) CHAPS 20 poly(methacrylic acid) Ficollpolyethyleneimine 21 poly(methacrylic acid) Ficoll Tween 22poly(methacrylic acid) Ficoll Triton 23 poly(methacrylic acid) FicollPluronic 24 poly(methacrylic acid) Ficoll CHAPS 25 poly(methacrylicacid) polyacrylamide polyethyleneimine 26 poly(methacrylic acid)polyacrylamide poly(propylene glycol) 27 poly(methacrylic acid)polyacrylamide Zonyl 28 poly(methacrylic acid) polyacrylamide Tween 29poly(methacrylic acid) polyacrylamide Triton 30 poly(methacrylic acid)polyacrylamide Pluronic 31 poly(methacrylic acid) polyacrylamideN,N-dimethyldodecylamine N-oxide 32 poly(methacrylic acid)polyacrylamide CHAPS 33 poly(methacrylic acid) polyethyleneiminecarboxy-polyacrylamide 34 poly(methacrylic acid) polyethyleneimine1-O-Octyl-B-D-glucopyranoside 35 poly(methacrylic acid)polyethyleneimine Pluronic 36 poly(methacrylic acid) polyethyleneimineCHAPS 37 poly(methacrylic acid) Pluronic F68 CHAPS 38 poly(acrylic acid)poly(ethylene glycol) polyacrylamide 39 poly(acrylic acid) poly(ethyleneglycol) poly(diallyldimethyl ammonium chloride 40 poly(acrylic acid)polyacrylamide Triton 41 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) 42 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)dextran 43 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) Ficoll 44poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) polyacrylamide 45poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) poly(diallyldimethylammonium chloride 46 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)dextran sulfate 47 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)chondroitin sulfate A 48 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)Tween 49 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) Pluronic 50poly(vinyl alcohol) poly(ethylene glycol) dextran 51 poly(vinyl alcohol)poly(ethylene glycol) Ficoll 52 poly(vinyl alcohol) poly(ethyleneglycol) polyacrylamide 53 poly(vinyl alcohol) poly(ethylene glycol)poly(diallyldimethyl ammonium chloride 54 poly(vinyl alcohol)poly(ethylene glycol) dextran sulfate 55 poly(vinyl alcohol)poly(ethylene glycol) Tween 56 poly(vinyl alcohol) dextran Ficoll 57poly(vinyl alcohol) dextran Tween 58 poly(vinyl alcohol) dextranPluronic 59 poly(vinyl alcohol) Ficoll Tween 60 poly(vinyl alcohol)Ficoll Pluronic 61 poly(vinyl alcohol) polyacrylamide Tween 62poly(vinyl alcohol) polyacrylamide Pluronic 63 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) dextran 64 poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) Ficoll 65 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyacrylamide 66 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)poly(diallyldimethyl ammonium chloride 67 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) dextran sulfate 68 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) polyethyleneimine 69 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) Tween 70 poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) 1-O-Octyl-B-D-glucopyranoside 71 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) CHAPS 72 poly(2-ethyl-2-oxazoline) dextran Ficoll73 poly(2-ethyl-2-oxazoline) dextran Tween 74 poly(2-ethyl-2-oxazoline)dextran Triton 75 poly(2-ethyl-2-oxazoline) dextran Pluronic 76poly(2-ethyl-2-oxazoline) dextran CHAPS 77 poly(2-ethyl-2-oxazoline)Ficoll polyethyleneimine 78 poly(2-ethyl-2-oxazoline) Ficoll Brij 79poly(2-ethyl-2-oxazoline) Ficoll Tween 80 poly(2-ethyl-2-oxazoline)Ficoll Triton 81 poly(2-ethyl-2-oxazoline) Ficoll Pluronic 82poly(2-ethyl-2-oxazoline) Ficoll CHAPS 83 poly(2-ethyl-2-oxazoline)polyacrylamide polyethyleneimine 84 poly(2-ethyl-2-oxazoline)polyacrylamide Tween 85 poly(2-ethyl-2-oxazoline) polyacrylamide Triton86 poly(2-ethyl-2-oxazoline) polyacrylamide Pluronic 87poly(2-ethyl-2-oxazoline) polyacrylamide CHAPS 88poly(2-ethyl-2-oxazoline) dextran sulfate poly(styrene sulfonic acid) 89poly(2-ethyl-2-oxazoline) polyethyleneimine1-O-Octyl-B-D-glucopyranoside 90 poly(2-ethyl-2-oxazoline)polyethyleneimine Pluronic 91 poly(2-ethyl-2-oxazoline)polyethyleneimine CHAPS 92 poly(2-ethyl-2-oxazoline) Pluronic F68 CHAPS93 poly(ethylene glycol) dextran Ficoll 94 poly(ethylene glycol) dextranpolyvinylpyrrolidone 95 poly(ethylene glycol) dextran Tween 96poly(ethylene glycol) dextran CHAPS 97 poly(ethylene glycol) Ficollpolyethyleneimine 98 poly(ethylene glycol) Ficoll Tween 99 poly(ethyleneglycol) Ficoll CHAPS 100 poly(ethylene glycol) polyacrylamidepolyethyleneimine 101 poly(ethylene glycol) polyacrylamide Tween 102poly(ethylene glycol) polyacrylamide CHAPS 103 poly(ethylene glycol)polyethyleneimine 1-O-Octyl-B-D-glucopyranoside 104 poly(ethyleneglycol) polyethyleneimine CHAPS 105 dextran Ficoll hydroxyethylcellulose 106 dextran Ficoll Tween 107 dextran Ficoll Triton 108 dextranFicoll Pluronic 109 dextran Ficoll CHAPS 110 dextranpolyvinylpyrrolidone poly(2-acrylamido-2-methyl-1-propanesulfonic acid)111 dextran hydroxyethyl cellulose Tween 112 dextran hydroxyethylcellulose Triton 113 dextran Pluronic F68 CHAPS 114 Ficollpolyethyleneimine Pluronic 115 Ficoll polyethyleneimine CHAPS 116 Ficollhydroxyethyl cellulose Tween 117 Ficoll hydroxyethyl cellulose Triton118 Ficoll Pluronic F68 CHAPS 119 polyacrylamide polyethyleneiminePluronic 120 polyacrylamide polyethyleneimine CHAPS 121 polyacrylamidePluronic F68 CHAPS 122 polyethyleneimine Pluronic F68 CHAPS 123 PEOZ PEGPVPNO 124 PEOZ PEI PVPNO 125 PEOZ PA PVPNO 126 PEOZ PMAA PVPNO 127 PEGPEI PVPNO 128 PEG PMAA PVPNO 129 PEG PA PVPNO 130 PEI PA PVPNO 131 PEIPMAA PVPNO 132 PA PMAA PVPNO 133 PEOZ PEG PVPNO 134 PEOZ TWEEN PVPNO 135PEOZ PA PVPNO 136 PEOZ PMAA PVPNO 137 PEG TWEEN PVPNO 138 TWEEN PA PVPNO139 TWEEN PMAA PVPNO 140 PA PMAA PVPNO 141 PEG PA PVPNO 142 PEG PMAAPVPNO

In yet another aspect, an aqueous four-phase system is described,comprising:

a phase-separated solution comprising four aqueous phases, wherein eachof the phases comprises a phase component and has a different densityand the phases, taken together, represent a density gradient, andwherein the phase component combination of the four phases selected fromthe group consisting of:

Number Phase component combinations 1 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Ficoll 2poly(methacrylic acid) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyacrylamide 3 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) poly(diallyldimethyl ammonium chloride 4poly(methacrylic acid) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyethyleneimine 5 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) Tween 20 6 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol)1-O-Octyl-B-D-glucopyranoside 7 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) CHAPS 8 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) Ficoll polyethyleneimine 9poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Ficoll Tween 10poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Ficoll Triton 11poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Ficoll Pluronic 12poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Ficoll CHAPS 13poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyacrylamidepolyethyleneimine 14 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)polyacrylamide Tween 15 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)polyacrylamide Triton 16 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) polyacrylamide Pluronic 17 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) polyacrylamide CHAPS 18 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) polyethyleneimine1-O-Octyl-B-D-glucopyranoside 19 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) polyethyleneimine Pluronic 20 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) polyethyleneimine CHAPS 21poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Pluronic F68 CHAPS 22poly(methacrylic acid) poly(ethylene glycol) Ficoll polyethyleneimine 23poly(methacrylic acid) poly(ethylene glycol) Ficoll Tween 24poly(methacrylic acid) poly(ethylene glycol) Ficoll CHAPS 25poly(methacrylic acid) poly(ethylene glycol) polyacrylamidepolyethyleneimine 26 poly(methacrylic acid) poly(ethylene glycol)polyacrylamide Tween 27 poly(methacrylic acid) poly(ethylene glycol)polyacrylamide CHAPS 28 poly(methacrylic acid) poly(ethylene glycol)polyethyleneimine 1-O-Octyl-B-D-glucopyranoside 29 poly(methacrylicacid) poly(ethylene glycol) polyethyleneimine CHAPS 30 poly(methacrylicacid) Ficoll polyethyleneimine Pluronic 31 poly(methacrylic acid) Ficollpolyethyleneimine CHAPS 32 poly(methacrylic acid) Ficoll Pluronic F68CHAPS 33 poly(methacrylic acid) polyacrylamide polyethyleneiminePluronic 34 poly(methacrylic acid) polyacrylamide polyethyleneimineCHAPS 35 poly(methacrylic acid) polyacrylamide Pluronic CHAPS 36poly(methacrylic acid) polyethyleneimine Pluronic CHAPS 37 poly(vinylalcohol) poly(2-ethyl-2-oxazoline) poly(ethylene glycol) dextran 38poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)Ficoll 39 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) polyacrylamide 40 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) poly(diallyldimethyl ammonium chloride 41poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)dextran sulfate 42 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) Tween 43 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) dextran Ficoll 44 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) dextran Tween 45 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) dextran Pluronic 46 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) Ficoll Tween 47 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) Ficoll Pluroni 48 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) polyacrylamide Tween 49 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) polyacrylamide Pluronic 50 poly(vinyl alcohol)poly(ethylene glycol) dextran Ficoll 51 poly(vinyl alcohol)poly(ethylene glycol) dextran Tween 52 poly(vinyl alcohol) poly(ethyleneglycol) Ficoll Tween 53 poly(vinyl alcohol) poly(ethylene glycol)polyacrylamide Tween 54 poly(vinyl alcohol) dextran Ficoll Tween 55poly(vinyl alcohol) dextran Ficoll Pluronic 56 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) dextran Ficoll 57 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) dextran Tween 58 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) dextran CHAPS 59 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) Ficoll polyethyleneimine 60poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Ficoll Tween 61poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Ficoll CHAPS 62poly(2-ethyl-2-oxazoline) poly(ethylene glycol) polyacrylamidepolyethyleneimine 63 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyacrylamide Tween 64 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyacrylamide CHAPS 65 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyethyleneimine 1-O-Octyl-B-D-glucopyranoside 66poly(2-ethyl-2-oxazoline) poly(ethylene glycol) polyethyleneimine CHAPS67 poly(2-ethyl-2-oxazoline) dextran Ficoll Tween 68poly(2-ethyl-2-oxazoline) dextran Ficoll Triton 69poly(2-ethyl-2-oxazoline) dextran Ficoll Pluronic 70poly(2-ethyl-2-oxazoline) dextran Ficoll CHAPS 71poly(2-ethyl-2-oxazoline) dextran Pluronic F68 CHAPS 72poly(2-ethyl-2-oxazoline) Ficoll polyethyleneimine Pluronic 73poly(2-ethyl-2-oxazoline) Ficoll polyethyleneimine CHAPS 74poly(2-ethyl-2-oxazoline) Ficoll Pluronic F68 CHAPS 75poly(2-ethyl-2-oxazoline) polyacrylamide polyethyleneimine Pluronic 76poly(2-ethyl-2-oxazoline) polyacrylamide polyethyleneimine CHAPS 77poly(2-ethyl-2-oxazoline) polyacrylamide Pluronic F68 CHAPS 78poly(2-ethyl-2-oxazoline) polyethyleneimine Pluronic F68 CHAPS 79poly(ethylene glycol) dextran Ficoll Tween 80 poly(ethylene glycol)dextran Ficoll CHAPS 81 poly(ethylene glycol) Ficoll polyethyleneimineCHAPS 82 poly(ethylene glycol) polyacrylamide polyethyleneimine CHAPS 83dextran Ficoll hydroxyethyl cellulose Tween 84 dextran Ficollhydroxyethyl cellulose Triton 85 dextran Ficoll Pluronic CHAPS 86 Ficollpolyethyleneimine Pluronic CHAPS 87 polyacrylamide polyethyleneiminePluronic CHAPS 88 PEOZ PEG PEI PVPNO 89 PEOZ PEG PA PVPNO 90 PEOZ PEI PAPVPNO 91 PEOZ PEI PMAA PVPNO 92 PEOZ PA PMAA PVPNO 93 PEG PEI PA PVPNO94 PEG PEI PMAA PVPNO 95 PEG PA PMAA PVPNO 96 PEI PA PMAA PVPNO 97 PEOZPEG PMAA PVPNO 98 PEOZ PEG PA PVPNO 99 PEOZ PEG TWEEN PVPNO 100 PEOZTWEEN PA PVPNO 101 PEOZ TWEEN PMAA PVPNO 102 PEOZ PA PMAA PVPNO 103 PEGTWEEN PA PVPNO 104 PEG TWEEN PMAA PVPNO 105 PEG PA PMAA PVPNO 106 TWEENPA PMAA PVPNO

In yet another aspect, an aqueous five-phase system is described,comprising:

a phase-separated solution comprising five aqueous phases, wherein eachof the phases comprises a phase component and has a different densityand the phases, taken together, represent a density gradient, andwherein the phase component combination of the five phases is selectedfrom the group consisting of:

number Phase component combinations 1 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) dextran Ficoll 2poly(methacrylic acid) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)Ficoll polyethyleneimine 3 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) polyacrylamidepolyethyleneimine 4 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) dextran Tween 5 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Ficoll Tween 6poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)Ficoll Tween 7 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) dextranFicoll Tween 8 poly(vinyl alcohol) poly(ethylene glycol) dextran FicollTween 9 poly(2-ethyl-2-oxazoline) poly(ethylene glycol) dextran FicollTween 10 poly(methacrylic acid) poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) polyacrylamide Tween 11 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) polyacrylamide Tween 12poly(methacrylic acid) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyethyleneimine 1-O-Octyl-B-D- glucopyranoside 13 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) dextran Ficoll Pluronic 14 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) Ficoll polyethyleneimine Pluronic 15poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyacrylamidepolyethyleneimine Pluronic 16 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Ficoll CHAPS 17poly(2-ethyl-2-oxazoline) poly(ethylene glycol) dextran Ficoll CHAPS 18poly(methacrylic acid) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyacrylamide CHAPS 19 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) polyethyleneimine CHAPS 20 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) Ficoll polyethyleneimine CHAPS 21poly(methacrylic acid) poly(ethylene glycol) Ficoll polyethyleneimineCHAPS 22 poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Ficollpolyethyleneimine CHAPS 23 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) polyacrylamide polyethyleneimine CHAPS 24poly(methacrylic acid) poly(ethylene glycol)′ polyacrylamidepolyethyleneimine CHAPS 25 poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) polyacrylamide polyethyleneimine CHAPS 26 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) Ficoll Pluronic CHAPS 27poly(2-ethyl-2-oxazoline) dextran Ficoll Pluronic CHAPS 28poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyacrylamide PluronicCHAPS 29 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)polyethyleneimine Pluronic CHAPS 30 poly(methacrylic acid) Ficollpolyethyleneimine Pluronic CHAPS 31 poly(2-ethyl-2-oxazoline) Ficollpolyethyleneimine Pluronic CHAPS 32 poly(methacrylic acid)polyacrylamide polyethyleneimine Pluronic CHAPS 33poly(2-ethyl-2-oxazoline) polyacrylamide polyethyleneimine PluronicCHAPS 34 PEG PEI PA PMAA PVPNO 35 PEOZ PEI PA PMAA PVPNO 36 PEOZ PEG PAPMAA PVPNO 37 PEOZ PEG PEI PMAA PVPNO 38 PEOZ PEG PEI PA PVPNO 39 PEOZPEG PEI PA PMAA 40 PEG TWEEN PA PMAA PVPNO 41 PEOZ TWEEN PA PMAA PVPNO42 PEOZ PEG TWEEN PMAA PVPNO 43 PEOZ PEG TWEEN PA PVPNO 44 PEOZ PEGTWEEN PA PMAA

In yet another aspect, an aqueous six-phase system is described,comprising:

a phase-separated solution comprising six aqueous phases, wherein eachof the phases comprises a phase component and has a different densityand the phases, taken together, represent a density gradient, andwherein the phase component combination of the six phases is selectedfrom the group consisting of:

number Phase component combinations 1 poly(vinyl poly(2-ethyl-poly(ethylene dextran Ficoll Tween alcohol) 2-oxazoline) glycol) 2 PMAAPEOZ PEG Ficoll PEI CHAPS 3 PMAA PEOZ PEG PA PEI CHAPS 4 PMAA PEOZ PEIFicoll CHAPS Pluronic 5 PMAA PEOZ PA PEI Pluronic CHAPS 6 PEOZ PEG PEIPA PMAA PVPNO 7 PEOZ PEG TWEEN PA PMAA PVPNO

In yet another aspect, a non-aqueous two-phase system is described,comprising:

two phase-separated non-aqueous phases, wherein each of the phasescomprises a phase component and has a different density and the phases,taken together, represent a density gradient, and wherein the phasecomponent combination of the two phases is selected from the groupconsisting of:

-   poly(bisphenol A carbonate)-polydimethylsiloxane, poly(bisphenol A    carbonate)-polystyrene,-   poly(bisphenol A carbonate)-poly(4-vinylpyridine), poly(bisphenol A    carbonate)-poly(2-ethyl-2-oxazoline), poly(bisphenol A    carbonate)-polycaprolactone, polydimethylsiloxane-polystyrene,    polydimethylsiloxane-poly(4-vinylpyridine),    polydimethylsiloxane-poly(2-ethyl-2-oxazoline),    polydimethylsiloxane-polycaprolactone,    polystyrene-poly(4-vinylpyridine),-   polystyrene-poly(2-ethyl-2-oxazoline), polystyrene-polycaprolactone,    poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),    poly(4-vinylpyridine)-polycaprolactone,    poly(2-ethyl-2-oxazoline)-polycaprolactone, PPG-PEG, PPG-PL;    PPG-PDMS; PPG-PBD; PEG-PL; PEG-PDMS; PEG-PEVE; PEG-PBD; PL-PDMS;    PL-PBD; PDMS-PEVE; PDMS-PBD; and PEVE-PBD.

In yet another aspect, a non-aqueous three-phase system is described,comprising:

three phase-separated non-aqueous phases, wherein each of the phasescomprises a phase component and has a different density and the phases,taken together, represent a density gradient, and wherein the phasecomponent combination of the three phases is selected from the groupconsisting of:

-   poly(bisphenol A carbonate)-polydimethylsiloxane-polystyrene,    poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(4-vinylpyridine),    poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(2-ethyl-2-oxazoline,    poly(bisphenol A carbonate)-polydimethylsiloxane-polycaprolactone,    poly(bisphenol A carbonate)-polystyrene-poly(4-vinylpyridine),    poly(bisphenol A carbonate)-polystyrene-poly(2-ethyl-2-oxazoline),    poly(bisphenol A carbonate)-polystyrene-polycaprolactone,    poly(bisphenol A    carbonate)-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),    poly(bisphenol A carbonate)-poly(4-vinylpyridine)-polycaprolactone,    poly(bisphenol A    carbonate)-poly(2-ethyl-2-oxazoline)-polycaprolactone, poly    dimethylsiloxane-poly styrene-poly(4-vinylpyridine), poly    dimethylsiloxane-poly styrene-poly(2-ethyl-2-oxazoline),    polydimethylsiloxane-polystyrene-polycaprolactone,    polydimethylsiloxane-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),    poly dimethylsiloxane-poly(4-vinylpyridine)-poly caprolactone,    polydimethylsiloxane-poly(2-ethyl-2-oxazoline)-polycaprolactone,    polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),    polystyrene-poly(4-vinylpyridine)-polycaprolactone,    polystyrene-poly(2-ethyl-2-oxazoline)-polycaprolactone,    poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone.    PPG-PEG-PL; PPG-PDMS-PBD; PPG-PEG-PBD; PEG-PEVE-PBD; PEG-PEVE-PDMS;    PPG-PEG-PDMS; PEG-PL-PDMS; PEG-PL-PBD; PL-PDMS-PBD; and    PDMS-PEVE-PBD.

In yet another aspect, a non-aqueous four-phase system is described,comprising:

four phase-separated non-aqueous phases, wherein each of the phasescomprises a phase component and has a different density and the phases,taken together, represent a density gradient, and wherein the phasecomponent combination of the four phases is selected from the groupconsisting of:

-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-poly(4-vinylpyridine),    poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-poly(2-ethyl-2-oxazoline),-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-polycaprolactone,    poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),    poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(4-vinylpyridine)polycaprolactone,    poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(2-ethyl-2-oxazoline)-polycaprolactone,    poly(bisphenol A    carbonate)-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),    poly(bisphenol A    carbonate)-polystyrene-poly(4-vinylpyridine)-polycaprolactone,    poly(bisphenol A    carbonate)-polystyrene-poly(2-ethyl-2-oxazoline)-polycaprolactone,    poly(bisphenol A    carbonate)-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,    polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),    polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-polycaprolactone,    polydimethylsiloxane-polystyrene-poly(2-ethyl-2-oxazoline)-polycaprolactone,    polydimethylsiloxane-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,    polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,    PPG-PEG-PL-PDMS; PPG-PEG-PL-PBD; PPG-PEG-PDMS-PBD; PPG-PL-PDMS-PBD;    and PEG-PL-PDMS-PBD.

In yet another aspect, a non-aqueous five-phase system is described,comprising:

five phase-separated non-aqueous phases, wherein each of the phasescomprises a phase component and has a different density and the phases,taken together, represent a density gradient, and wherein the phasecomponent combination of the five phases is selected from the groupconsisting of:

poly(bisphenol Acarbonate)-polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),poly(bisphenol Acarbonate)-polydimethylsiloxane-polystyrene-polycaprolactone,poly(bisphenol Acarbonate)-polydimethylsiloxane-polystyrene-poly(2-ethyl-2-oxazoline)-polycaprolactone,poly(bisphenol. Acarbonate)-polydimethylsiloxane-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,poly(bisphenol Acarbonate)-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,and PPG-PEG-PL-PDMS-PBD.

In yet another aspect, a non-aqueous six-phase system is described,comprising:

a phase-separated solution comprising six non-aqueous phases, whereineach of the phases has a different density and the phases, takentogether, represent a density gradient, and wherein the composition ofthe phases is selected from the group consisting of:

-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone.

In yet another aspect, a kit is described, comprising:

a plurality of phase components, in one or more solvents or in theirsolvent-free forms, selected from the group consisting of a polymer, asurfactant and combinations thereof, wherein at least one phasecomponent comprises a polymer, each said phase component packagedseparately;

instructions for combining the plurality of phase components in one ormore solvents and for preparing a phase-separated solution therefrom;and

instructions for analyzing an analyte the phase-separated solution.

In yet another aspect, a method of analyzing or separating a sample isdescribed, comprising:

providing a phase-separated system comprising at least two phases,wherein

-   -   the at least two phases each comprises a phase component        selected from the group consisting of a polymer, a surfactant        and combinations thereof, wherein at least one phase comprises a        polymer;    -   each said phase has an upper and a lower phase boundary; and    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and

b) introducing a sample comprising one or more analytes of interest tothe multi-phase system without disrupting the phase-separated solution;and

c) allowing each of the analytes to migrate to a location in thephase-separated system that is characteristic of its density, whereinduring migration the sample contacts one or more of the two or morephases sequentially.

In any of the preceding embodiments, greater than 80% of the analyte islocated at one or more of the phase boundaries.

In any of the preceding embodiments, greater than 90% of the analyte islocated at one or more of the phase boundaries.

In any of the preceding embodiments, the sample comprises a plurality ofanalytes and each analyte migrates to a different location in thephase-separated system.

In any of the preceding embodiments, after migration, the analyteresides at a boundary location.

In any of the preceding embodiments, the boundary location is at aninterface between a phase with a density greater than the density of theanalyte and a phase with a density that is less than the density of theanalyte.

In any of the preceding embodiments, after migration, the analyteresides within a phase of the phase-separated system whose densitymatches the density of the analyte.

In any of the preceding embodiments, the analyte/phase-separated systemis centrifuged to accelerate migration of the analyte.

In any of the preceding embodiments, the analyte migrates undergravitational forces.

In any of the preceding embodiments, the analyte migrates under buoyancyforces.

In any of the preceding embodiments, the phase separated system issupported in a column or test tube.

In any of the preceding embodiments, the phase separated system issupported along a filament or on a sheet.

In any of the preceding embodiments, the multi-phases are provided asdispersion or emulsion in another carrier phase.

In any of the preceding embodiments, the analyte of interest a size ofmore than 200 nm.

In any of the preceding embodiments, after analyte migration the phasesand the analyte are in thermodynamic equilibrium.

In any of the preceding embodiments, the phase separated systemcomprises three or more phases.

In any of the preceding embodiments, the two or more phases comprise acommon solvent which is an aqueous solvent.

In any of the preceding embodiments, the two or more phases comprise acommon solvent which is an organic solvent.

In any of the preceding embodiments, the two or more phases comprise acommon solvent which is a non-aqueous solvent selected from the groupsconsisting of liquid polymer, non-polar organic solvent, polar aproticor protic solvent, supercritical fluid, fuel, oils, and fluorinatedsolvents, and combinations thereof.

In any of the preceding embodiments, the common solvent comprisesdichloromethane.

In any of the preceding embodiments, the phase separated systemcomprises three or more phases including the two or more phasescomprising a common solvent which is water and additional phaseseparated phases selected from the group consisting of organicsolutions.

In any of the preceding embodiments, the aqueous solvent is selectedfrom the group consisting of water, sea water, isotopes of water,buffered water, irrigation water, mine effluent, colloidal solutions,emulsions, and a combination thereof.

In any of the preceding embodiments, the analyte is selected from thegroup consisting of solid particles, an aggregate of particles, a liquidor gel immiscible in the solvent, a liquid crystal, crystallinematerials.

In any of the preceding embodiments, the analyte is selected from thegroup consisting of gem, bead, metal, glass, rock, mineral, crystal,plastic, bone, rubber, paper, fabric, coal, polymer particles, gases.

In any of the preceding embodiments, the polymer is selected from thegroup of homopolymers, random copolymers, block copolymers, graftcopolymers, ter-polymers, dendrimers, star polymers and combinationsthereof.

In any of the preceding embodiments, the polymer is linear, branchedand/or cross-linked.

In any of the preceding embodiments, the polymer is selected from thegroup consisting of dextran, dextran sulfate, chondroitin sulfate A,polysucrose, diethylaminoethyl-dextran, poly(2-vinylpyridine-N-oxide),polysucrose, poly(vinyl alcohol), poly(2-ethyl-2-oxazoline),poly(methacrylic acid), poly(ethylene glycol), polyacrylamide,polyethyleneimine, hydroxyethyl cellulose, polyvinylpyrrolidone,carboxy-polyacrylamide, poly(acrylic acid),poly(2-acrylamido-2-methyl-1-propanesulfonic acid), poly(diallyldimethylammonium chloride), poly(styrene sulfonic acid), polyallylamine, alginicacid, poly(bisphenol A carbonate), polydimethylsiloxane, polystyrene,poly(4-vinylpyridine), polycaprolactone, polysulfone, poly(methylmethacrylate-co-methacrylic acid), poly(methyl methacrylate),poly(tetrahydrofuran), poly(propylene glycol), and poly(vinyl acetate)and copolymers or terpolymer thereof.

In any of the preceding embodiments, the surfactant is selected from thegroup consisting of polysorbate,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate,polyoxyethylene-polyoxypropylene, 1-O-Octyl-β-D-glucopyranoside,Tetramethylbutyl)phenyl-polyethylene glycol, nonylphenolpolyoxyethylene, 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,2-(Perfluoroalkyl)ethyl methacrylate, N,N-dimethyldodecylamine N-oxide,polyethylene glycol dodecyl ether, sodium dodecyl sulfate, sodiumcholate, benzylalkonium chloride and dodecyltrimethylammonium chloride.

In any of the preceding embodiments, one or more phases further comprisean additive selected from the group consisting of a so-solvent, an acid,a base, a miscible polymer, vitamin, drug, antibiotic, small molecule,dye, and flurophore.

In any of the preceding embodiments, the sample is selected from thegroup consisting of forensics study sample, a sample indicative ofanimal health, a sample indicative of human identity used for bordercontrol, home land security, or intelligence, a sample from foodprocessing, a sample indicative of product quality, a sample indicativeof environmental safety, a sample containing different crystalpolymorphs, and a combination thereof.

In any of the preceding embodiments, the phases in the multi-phases havelow interfacial free energy.

In any of the preceding embodiments, the method further comprisescollecting the analyte from the boundary location.

In yet another aspect, a method of determining the density of a solidparticle using a multi-phase system is described, comprising:

a) providing a multi-phase system comprising two or more phases, wherein

-   -   at least two of the phases each comprise a phase component        selected from the group consisting of a polymer, a surfactant,        and a combination thereof and at least one of the two phase        comprises a polymer;    -   each of the two or more phases has a different density and the        two or more phases, taken together, represent a density        gradient; and    -   the phases are phase-separated from each other;

b) introducing a solid particle to the multi-phase system;

c) allowing the solid particle to migrate to a location in themultiphase system that is characteristic of its density, wherein duringmigration the sample contacts one or more of the two or more phasessequentially; and

d) determining the density of the solid particle, wherein the solidparticle resides in an interface between two adjacent phases and thedensity of the solid particle is calculated based on the position of thesolid particle, the buoyancy, and the interfacial tension between thetwo phases.

In any of the preceding embodiments, the solid particle's density isdetermined by using the equation that gravity of the solid particle(F_(g); N)=buoyancy (F_(B); N)+ the interfacial tension (F_(I); N).

In yet another aspect, a method of creating a multi-phase system isdescribed, comprising:

identifying a two-phase system comprising a first phase comprising afirst polymer and a second phase comprising a second polymer, whereinthe first and second phases are and phase-separated from each other; and

introducing a third phase comprising a copolymer of the first and secondpolymers to the two-phase system; wherein

the copolymer is formed by polymerization of the monomers of the firstand second polymers, and

the first, second, and third phase are phase-separated from each other.

In any of the preceding embodiments, the first, second, and third phasesare each organic or each aqueous.

In yet another aspect, a method of analyzing an analyte is described,comprising:

mixing a sample containing an analyte of interest and a plurality ofphase components in an appropriate solvent as set forth in themulti-phase system of any one of the preceding embodiments;

allowing the mixture to phase separate, wherein the analyte interactswith all of phase components during phase separate, wherein the analytepreferentially resides in one phase based on the analyte's affinity tothat phase.

In yet another aspect, a method of preparing a multi-phase system isdescribed, comprising:

mixing at least a substantially pure first and second phase componentsand a common solvent to form a mixture, wherein the first and secondphase components are each selected from the group consisting of apolymer, a surfactant, and a combination thereof and at least of thefirst and second phase components is a polymer; and

allowing the mixture to phase-separate to form a multi-phase systemcomprising at least:

-   -   a first phase comprising the first phase component; and    -   a second phase comprising the second phase component; wherein        the first and second phase are in contact and phase-separate        from each other.

In any of the preceding embodiments, the method further comprisesshaking or stirring the mixture.

In any of the preceding embodiments, the common solvent is aqueous ororganic.

In yet another aspect, a method of the broadening the density range ofan existing multi-phase system is described, comprising:

providing an existing multi-phase system comprising two or more phasesincluding at least a top phase and a bottom phase at the top and bottomof the existing multi-phase system, respectively, and a first and secondphases. wherein

-   -   the first and second phases are in contact, phase-separated from        each other, and each comprising a phase component selected from        the group consisting of a polymer, a surfactant, and a        combination thereof;    -   at least one of the first and second phases comprising a        polymer;    -   each of the two or more phases has a different density and the        two or more phases, taken together, represent a density        gradient;

adding, to the existing multi-phase system, one or more additionalphase(s) with densities lower than the top phase of the existingmulti-phase system; and

allowing the additional phase(s) and the existing multi-phase system tophase-separate to form a new multi-phase system.

In yet another aspect, a method of the broadening the density range ofan existing multi-phase system is described, comprising:

providing an existing multi-phase system comprising two or more phasesincluding at least a top phase and a bottom phase at the top and bottomof the existing multi-phase system, respectively, and a first and secondphases. wherein

-   -   the first and second phases share a common solvent,        phase-separated from each other, and each comprising a phase        component selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   at least one of the first and second phases comprising a        polymer;    -   each of the two or more phases has a different density and the        two or more phases, taken together, represent a density        gradient;

adding, to the existing multi-phase system, one or more additionalphase(s) with densities higher than the bottom phase of the existingmulti-phase system; and

allowing the additional phase(s) and the existing multi-phase system tophase-separate to form a new multi-phase system.

In any of the preceding embodiments, the additional phase is aqueous ororganic.

In any of the preceding embodiments, the additional phase comprises aphase component selected from the group consisting of a polymer, asurfactant, and a combination thereof.

In yet another aspect, a method of the shifting the density range of anexisting multi-phase system is described, comprising:

providing an existing multi-phase system comprising two or more phasescomprising a common solvent and including at least a first and secondphases, wherein

-   -   the first and second phases share a common solvent,        phase-separated from each other, and each comprising a phase        component selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   at least one of the first and second phases comprising a        polymer;    -   each of the two or more phases has a different density and the        two or more phases, taken together, represent a density        gradient;

adding, to the existing multi-phase system, one or more additive(s) withdensities higher or lower than the common solvent to form a mixture; and

allowing the mixture to phase-separate to form a new multi-phase systemwith a density range different from that of the existing multi-phasesystem.

In any of the preceding embodiments, the additive is a co-solventmixable with the common solvent.

In any of the preceding embodiments, the additive is a salt soluble inthe common solvent.

In any of the preceding embodiments, one or more phases are asolvent-free liquid polymer phase.

BRIEF DESCRIPTION OF THE DRAWING

The subject matter is described with reference to the following figures,which are presented for the purpose of illustration only and are notintended to be limiting of the invention.

FIG. 1 is plot of the miscibility characteristics of binary mixtures ofaqueous polymer solutions, in which light gray indicates miscible binarysystems, dark gray indicates immiscible binary systems and blackrepresents incompatible binary systems.

FIG. 2A-2C shows images of multiphase polymer systems. A) A three-phasesystem comprised of poly(propyleneglycol)-polyacrylamide-poly(methacrylic acid). B) A four-phase systemcomprised of poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethyleneglycol)-polyacrylamide. C) A five-phase system comprised of poly(vinylalcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran-Ficoll.

FIG. 3A-3B shows images of a single barrier separating density standardsbeads based on small differences in density.

FIG. 4 shows an image of dyed density standards banding at thepolymer/polymer interface of the triphasic poly(vinylalcohol)/poly(ethylene glycol)/dextran system.

FIG. 5 illustrates dichloromethane-polymer-polymer ternary mixtures,categorized as either biphasic (+), homogeneous (−), or incompatible(0).

FIG. 6 illustrates a two phase MPS used for separating beads based ondensity.

FIG. 7A-7B illustrates forces on a sphere at an interface. (A) An imageof a polystyrene bead (ρ=1.0456 g/cm³) at the interface between apoly(ethylene glycol)-Ficoll aqueous two-phase polymer system (layerdensities of 1.0320 g/cm³ and 1.1122 g/cm³, respectively). (B) Anoverlayed schematic of the three forces acting on the bead at theliquid/liquid interface: gravitational force (F_(g)), buoyant force(F_(B)), and interfacial tension (F_(I)).

FIG. 8 illustrates a density-based separation using a two-phase MPS.

FIG. 9 shows a schematic of the forces acting on a sphere at aninterface.

FIG. 10 A illustrates a two-phase MPS deposited on patterned-paper.

FIG. 10 B shows a selective accumulation of Allura Red in the top phaseof the PEG/PMAA two-phase system.

FIG. 11A(i) shows a patterned paper with Allura red spotted inhydrophilic regions.

FIG. 11A(ii) shows the top and bottom phases of a PEG-PMAA two-phase APSspotted on two sets of hydrophilic regions on a patterned paper.

FIG. 11B shows the selective accumulation of Allura Red in the top phaseof the PEG/PMAA two-phase system deposited on the hydrophilic regions ofthe patterned paper.

FIG. 12A shows a shaken mixture of mineral oil, ionic liquid, siliconeoil, perfluorohexane, mercury, and five aqueous phase component stocksolution.

FIG. 12B shows a 10-phase MPS including, from top to bottom, a mineraloil phase, a ionic liquid phase, a silicone oil phase, 5 aqueous phases,a perfluorohexane phase, and a mercury phase.

FIG. 13 shows a MPS's density range change with the addition of isotopicwater.

FIG. 14 shows a MPS's density range change with the addition of salt.

DETAILED DESCRIPTION Introduction

The disclosed methods are used to separate objects or impurities insamples according to the densities of the objects or impurities,relative to the densities of the phases of a MPS. Everything has adensity. Thus, because the disclosed methods can be used to separate,isolate, characterize, analyze, prepare, and purify such diverseobjects, the disclosed methods can be applied to many contexts. Forexample, the disclosed methods can be used in the forensics, security,and intelligence contexts to separate and process objects of interestfrom complex samples, e.g., to detect explosives residues or separatebiological specimens from samples that have been contaminated byenvironmental debris from crime scenes. These methods can also be usedmonitor animal and plant health. Animal tissues and plant material canbe broken down to the cellular level to detect cellular abnormalitiesindicative of disease and infection. Similarly, these methods can beused to detect contaminants such as pathogens, pests, heavy metals, andpesticides in drinking water and in food processing to ensure qualitycontrol.

Multi-Phase Systems

A multi-phase system comprising two or more phases is described, whereinthe two or more phases include at least a first and second phases incontact with and phase-separated from each other, each of first andsecond phases comprises a phase component selected from the groupconsisting of a polymer, a surfactant, and a combination thereof, and atleast one of the first and second phases comprise a polymer. Each of thetwo or more phases has a different density and the phases, takentogether, represent a density gradient, with the density of the phasesincreasing from the top phase to the bottom phase of the MPS.

In some embodiments, the MPS includes at least two phases with a commonsolvent. In some embodiments, the multi-phase polymer system comprisesat least three phases. In some embodiments, the multi-phase systemcomprises at least four phases. In some embodiments, the multi-phasepolymer system comprises at least five phases. In some embodiments, themulti-phase polymer system comprises at least six phases. Multi-phasesystem with more phases are contemplated. When more than two phases areused, it is possible to include phases using different solvents. It isalso possible to include phases that do not include a phase component,such as aqueous or organic solvents, liquid polymers, fluorinatedliquids, liquid metals, e.g., mercury, and ionic liquids. Such varietyimproves the ability of the system to separate complex samples. Forexample, the additional phases can extend the density range of thesample, making it possible to separate or distinguish samples of higheror lower density. Non-limiting examples of liquid polymers includepoly(propylene glycol) (PPG), poly(ethylene glycol) (PEG), Pluronic L121(PL), polydimethylsiloxane (PDMS), poly(ethyl vinyl ether) (PEVE),polybutadiene (PBD).

Each of the phases of the multi-phase system comprises a phasecomponent. The phase component is selected from the group consisting ofa polymer, a surfactant, and combinations thereof.

An exemplary MPS system is shown in FIG. 4. The flexible tubing containsa 3-phase multi-phase system prepared from poly(vinyl alcohol) (“PVA”)(ρ=1.056 g/cm³), poly(ethylene glycol) (“PEG”) (ρ=1.063) and dextran(ρ=1.152 g/cm³), all in water. PVA, the polymer of lowest density,represents the ‘top’ of the density gradient and dextran, the lowest.The delineation of each band can be observed from the location ofcolored density standard beads. Orange beads having an intermediatedensity between that of PVA and PEG are located at the PVA/PEGinterface. Green beads having an intermediate density between that ofPEG and dextran are located at the PEG/dextran interface. Purple beadsthat are denser than dextran sit below the dextran band. This exampledemonstrates the ability of the multi-phase system to distinguishbetween phases density variations of as little as ±0.01 g/cm³.Distinctions of as little as ±0.001 g/cm³ have been demonstrated.

Non-limiting examples of polymer include dextran, polysucrose (hereinreferred to by the trade name “Ficoll”), poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), poly(methacrylic acid), poly(ethyleneglycol), polyacrylamide, polyethyleneimine, hydroxyethyl cellulose,polyvinylpyrrolidone, carboxy-polyacrylamide, poly(acrylic acid),poly(2-acrylamido-2-methyl-1-propanesulfonic acid), dextran sulfate,diethylaminoethyl-dextran, chondroitin sulfate A,poly(2-vinylpyridine-N-oxide), poly(diallyldimethyl ammonium chloride),poly(styrene sulfonic acid), polyallylamine, alginic acid, nonylphenolpolyoxyethylene, poly(bisphenol A carbonate), polydimethylsiloxane,polystyrene, poly(4-vinylpyridine), polycaprolactone, polysulfone,poly(methyl methacrylate-co-methacrylic acid), poly(methylmethacrylate), poly(tetrahydrofuran), poly(propylene glycol), andpoly(vinyl acetate). As used herein, a polymer includes its homopolymer,copolymer, terpolymer, block copolymer, random polymer, linear polymer,branched polymer, crosslinked polymer, and/or dendrimer system.

Non-limiting examples of surfactants include polysorbate (hereinreferred to by the trade name “Tween”),3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),polyoxyethylene-polyoxypropylene (herein referred to by the trade name“Pluronic”), 1-O-Octyl-β-D-glucopyranoside,4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (herein referredto by the trade name “Triton”), 2-(Perfluoroalkyl)ethyl methacrylate(herein referred to by the trade name “Zonyl”), N,N-dimethyldodecylamineN-oxide, polyethylene glycol dodecyl ether (herein referred to by thetrade name “Brij”), sodium dodecyl sulfate, sodium cholate,benzylalkonium chloride and dodecyltrimethylammonium chloride. In somespecific embodiments, surfactant phases comprising Pluronic and CHAPSare selected to form an aqueous multi-phase polymer system with one ormore aqueous polymer phases. Non-limiting examples of the polymer usedin these embodiments include poly(methacrylic acid),poly(2-ethyl-2-oxazoline), dextran, Ficoll, polyacrylamide, andpolyethyleneimine. The use of the surfactant can provide additionalaqueous phases and facilitate the formation of the multi-phase systems.Other appropriate surfactants to accomplish this objective can beselected by the persons of ordinary skills in the art.

The phase components are selected so that the resulting phases arephase-separated from each other. As used herein, phase-separation refersto the phenomena where two ore more solutions, each comprising a phasecomponent, when mixed together, form the same number of distinct phaseswhere each phase has clear boundaries and is separated from otherphases. Each phase component used in the solution is selected to besoluble in the solvent of the phase, so that each resulting phase is adistinct solution of the phase component and that each phase isphase-separated from other adjacent phase(s). When the multi-phasepolymer system is designed, each phase component is selected topredominantly reside in one particular phase of the multi-phase system.It should be noted that in the resulting multi-phase system, every phasecan contain varying amounts of other phase components from other phasesin the MPS, in addition to the selected desired phase component in thatphase. Unless otherwise specified, the phase component composition ineach phase of the multi-phase system recited herein generally refers tothe starting phase component composition of each phase, or to thepredominant phase component composition of each phase. In someembodiments, the phase component composition on a phase componentcomprises predominantly one phase component and small amount of one ormore other phase components. In some embodiments, the phase componentcomposition in a phase comprises more than 50% and more typically morethan about 70% of one phase component. In some embodiments, the phasecomponent composition in a polymer phase comprises more than about 75%of one phase component. In some embodiments, the phase componentcomposition in a phase comprises more than about 80% of one phasecomponent. In some embodiments, the phase component composition on aphase comprises more than about 85% of one phase component by weight. Insome embodiments, the phase component composition on a phase comprisesmore than about 90% of one phase component by weight. In someembodiments, the phase component composition in a phase comprises morethan about 95% of one phase component. In some embodiments, the phasecomponent composition in a phase comprises more than about 99% of onephase component by weight. Other combinations of phase componentcompositions are contemplated.

In some embodiments, the concentration of the phase component in thephase is from about 0.1% to about 50% (wt/vol). In some embodiments, theconcentration of the phase component in the phase is from about 0.5% toabout 40% (wt/vol). In some embodiments, the concentration of the phasecomponent in the phase is from about 1% to about 20% (wt/vol). In someembodiments, the concentration of the phase component in the phase isfrom about 5% to about 10% (wt/vol). In some embodiments, theconcentration of the phase component in the phase is about 10% (wt/vol).In some embodiments, the concentration of the phase component in thephase is about 15% (wt/vol). In some embodiments, the composition ordensity of the resulting phases in the multi-phase system could beaffected by the starting concentration of the phase component phases.

In some embodiments, the multi-phase system is aqueous and each phase ofthe MPS comprises a phase component soluble in an aqueous solvent.Non-limiting examples of aqueous solvent include water, D₂O, seawater,mine effluent, and irrigation water and mixtures thereof. Seawater couldbe used in studying small ocean organisms to keep buoyant densitiesclose to what they are in nature. Irrigation water or mine effluentcould be used to study the density effects on micro-organisms whenexposed to these liquids. The analytes may also include particulatematter that could be suspended in these aqueous solutions.

In some embodiments, the aqueous multi-phase systems can compriseadditional one or more organic phases comprising organic solvents. Theorganic phase is immiscible with and phase-separated from the aqueouspolymer phases. Such additional phases are not required to have a phasecomponent.

In some embodiments described herein, the multi-phase may comprise atleast one aqueous phase. In some embodiments described herein, themulti-phase may comprise at least one organic phase. In some embodimentsdescribed herein, the multi-phase may comprise all organic phases or allaqueous phases.

In some embodiments, the aqueous multi-phase systems may be combinedwith one or more phases comprising organic solvents. Suitable organicsolvents are those that are immiscible with water and willphase-separate from the aqueous phases.

In some embodiments, the multi-phase system is organic and each phase ofthe MPS comprises a phase component dissolved in an organic solvent.

In some specific embodiments, the different phases of the MPS comprisethe same organic solvent. In other specific embodiments, the differentphases of the MPS comprise different organic solvent.

Non-limiting examples of organic solvent include organic solventselected from the groups consisting of liquid polymer, non-polar organicsolvent, polar aprotic or protic solvent. Non-limiting examples ofnon-polar organic solvent include hexane and xylene. Non-limitingexamples of polar aprotic organic solvent include dichloromethane andchloroform. Non-limiting examples of polar protic solvent includeethanol and methanol. Other suitable examples of organic solventsinclude supercritical fluid, fuel, oils, and fluorinated solvents, andcombinations thereof. In some embodiments, non-limiting examples ofsuitable organic solvents include chloroform, dichloromethane ether,ethyl acetate, dimethylformamide, benzene, toluene, xylene, hexanes,acetonitrile, diethylether, trichloroethane, benzyl alcohol, acetone,aniline, mineral oil, perfluorinated solvents, and oils, tetrahydrofuran(THF), or water miscible solvents such as ethanol and methanol,supercritical CO₂, complex hydrocarbons such as fuel, and hydrophobic,high viscosity fluids such as lubricants. For instance, using fuel as acommon solvent for a MPS, particulates suspended in fuel can be analyzedthat are the result from engine breakdown, fuel contamination,shale/rock particulates (from freshly mined fuels), etc.

In other embodiments, the MPS comprises a liquid polymer as one of thephases. Non-limiting examples of liquid polymers includepolyethyleneimine, polybutadiene, polydimethylsiloxane, poly(propyleneglycol), poly(ethyl vinyl ether), cis(polyisoprene) and Tween(surfactant). Such additional phases are not required to have a phasecomponent.

In some embodiments, the multi-phase system comprises at least anaqueous phase and at least an organic phase. Each phase may comprise aphase component and the mixture of aqueous and organic phases, takentogether, represents a density gradient.

In some embodiments, one or more of the phases of the MPS are degassedto remove residual amount of gas dissolved in the phases. In someembodiments, the phases are degassed to remove oxygen from the phase toavoid possible oxidation of the sample applied onto the MPS. Forexample, highly viscous phases of MPS are degassed to remove any bubblesentrapped in phases or at interfaces. Degassing can also remove H₂, N₂,CH₄, NH₃, Ar, and other trace gases such as H₂S, and NOx. Other gasesmay be added (e.g., ammonia), to perform chemistry on separated species.

In some embodiments, one or more salt can be added to an aqueousmulti-phase phase component system. The salts dissolve in the phaseresulting in change of the phase density and typically do not partitionbetween phases. Salts can also change the ionic strength of thesolution. Non-limiting examples of salts include light or heavy salts,NaCl, NaBr, LiBr, KBr, RbBr, CsBr, and some phosphate salts. Other saltscould be sodium metatungstate or manganese chloride. Non-limitingexamples of salts also include sodium chloride, potassium chloride,sulfates, phosphates, nitrites, and citrates. The addition of salts canhelp the phase-separation process. In some embodiments, salt(s) can beadded to the phase component systems in order to adjust the density, pH,and/or osmolality of the multiphase systems. In some embodiments, smallmolecules can be added for some specific functions. In some specificembodiments, heparin or sodium EDTA is added as an anticoagulant. Insome other embodiments, sodium benzoate is added as a preservative. Inother embodiments, paramagnetic salts are added to exploit magneticproperties.

In one or more embodiments, particularly multi-phase systems designedfor use with more than two phase components, one or more polymers orsurfactants that do not phase separate with each of the other phasecomponents can be used as additives to modify the density, viscosity,osmolality, or refractive index of the phase component in which theadditive resides. The polymers or surfactants are added to the variousphases of the multi-phase system in addition to the phase components atconcentrations less than is required to phase separate into a separatephase. In this instance, the surfactant performs the functions that aretypical of surfactants, such as modify the surface tension of thesolution.

Non-limiting examples of other additives that can be included in thephases include used in formulations to produce aggregation include,organic additives such as dyes and reactive or non-reactive dissolvedgasses and cosolvents. In addition, the phases can be colloids ormicelles.

Various types of form factors of the MPS can be used. In someembodiments, the MPS is contained in a tube or container, such as a testtube or flexible plastic tubing. In still other embodiments, the MPS isdeposited on cloth or string. In still other embodiments, the MPS isdeposited in bottle or drum or on porous films or sponges. For example,a string or porous filament can be held in a test tube during theformation of the multi-phase system, such that the phase separateddomains are absorbed into the porous filament. The filament is thenremoved from the MPS and contains a thin layer of phase-separateddomains along the length of the filament.

In some embodiments, the MPS is deposited on paper. In some embodiments,the MPS is deposited on patterned paper. Paper can be patterned usinghydrophobic barrier substantially permeating the thickness of the paper,thus defining one or more hydrophilic regions on the paper. In somespecific embodiments, the paper can be patterned following theprocedures described in PCT Publication No. 2008/049083, the content ofwhich is incorporated in its entirety by reference. In some specificembodiments, the MPS is aqueous and the aqueous phases of the MPS aredeposited on a plurality of the hydrophilic regions on paper. By way ofexample, phase separation bands from an MPS column can be individuallyspotted on patterned regions of paper. The individual spotted regionscan be stacked to recreate the density gradient in the MPS column.Alternatively, the individual spotted regions can be stacked to providea density variation that is different from the original MPS column.

In some embodiments, the MPSs as described herein have thecharacteristics that, once formed, are at equilibrium: (i) thecompositions and properties of the phase-separated layers do not changewith time, (ii) MPS may be prepared well in advance of their use, and(iii) MPS may be reformed if perturbed or agitated.

Generally speaking, if a combination of multiple phase component phasesresults in a phase-separated MPS, any sub-combination of the multiplephase component phases will also result in a phase-separated MPS. Thus,if a five-phase component MPS phase-separates, any four-polymer aqueoussystem selected from the five phase component phases can alsophase-separate. Likewise, any two- or three-phase component MPS selectedfrom the five phase component phases can also phase-separate. Othersuitable combinations of polymers are contemplated.

In some embodiments, whether or not a MPS comprising multiple phasecomponents will phase-separate can be predicted based on the propertiesof the MPSs containing the sub-combination of the multiple phasecomponents. For instance, phase solutions containing phase components A,B, and C, respectively, will phase-separate and form a three-phase MPSif the phase component A solution and phase component B solutionphase-separate, the phase component A solution and phase component Csolution phase-separate, the phase component B solution and phasecomponent C solution phase-separate. Similarly, solutions of phasecomponents A, B, C, and D will form a four-phase MPS if the followingphase components combinations all phase-separate: A-B-C, A-B-D, A-C-D,and B-C-D. Likewise, solutions of phase components A, B, C, D, and Ewill form a five-phase MPS if the following phase componentscombinations all phase-separate: A-B-C-D, A-B-C-E, A-B-D-E, A-C-D-E,B-C-D-E. Also, solutions of phase components A, B, C, D, E, and F willform a six-phase MPS if the following phase components combinations allphase-separate: A-B-C-D-E, A-B-C-D-F, A-B-C-E-F, A-B-D-E-F, A-C-D-E-F,and B-C-D-E-F. The prediction of more complex MPSs based on the sameprinciple is contemplated. These predictions have largely been confirmedby experimental data. Certain predicted MPS have not been produced byexperiments can be produced via routine experimental optimization.

As used herein, a MPS can be identified by its phase components in thephases of the MPS. For instance, aFicoll-dextran-poly(2-ethyl-2-oxazoline) system refers to a three-phaseMPS, wherein the phase components in each phases of the MPS are Ficoll,dextran, and poly(2-ethyl-2-oxazoline), but not necessarily in thatorder. Each phase includes a suitable solvent capable of dissolving thephase components. In some instances, a liquid polymer is used and theliquid polymer forms a phase with no solvent added.

Method

1. Method of Preparing a Multi-Phase System

Multi-phase systems can be prepared by preparing stock solutions of thedesired phase components, mixing the desired stock solutions, andallowing the mixture to phase separate. Appropriate volumes of stocksolutions are combined. Salts can be added to facilitate polymer-polymerphase separation. Typically, the combined stock solutions arecentrifuged to accelerate phase separation. Although separation canoccur by gravity settling, the time scales are longer thancentrifugation.

Alternatively, MPSs can be prepared by combined the desired phasecomponents in their substantially pure solid or liquid form with acommon solvent, followed by mixing or shaking the mixture and allowingthe mixture to phase separate. As used herein, a substantially purephase component refers to a polymer or surfactant with more than 80%,85%, 90%, 95%, 98%, 99%, or 99.9% purity and free of any solvent. Acombination of these two methods can also be used, e.g., one or morephase component stock solutions can be mixed with one or more phasecomponents in their pure solid or liquid forms, and the mixture can thenbe mixed or shaken and allowed to phase separate. In an exemplaryembodiment, stock solutions of 1 mL 20% PVA, 1 mL 20% PEG, and 1 mL 40%dextran can be combined and allowed to phase separate to form athree-phase MPS. Alternatively, 200 mg PVA, 200 mg PEG, 400 mg dextran,and 3 mL water can be combined and allowed to phase separate to form thesame three-phase MPS.

In yet another aspect, a multi-phase system can be prepared based on theknown behavior of existing multiphase systems. Once a two-phase systemis identified, having a first phase with a first polymer (A) and asecond phase with a second polymer (B), it is possible to prepare athree phase polymer system by introducing a third phase having acopolymer of the first and second polymers to the two-phase system,e.g., the copolymer is an A-B block, an A-B-A block, etc. In otherwords, once two polymers are identified that form immiscible phases,then it is expected that copolymers of these two immiscible polymerswill phase separate from both starting homopolymers. Non-limitingexamples of the first and the second polymers include poly(ethyleneglycol)-poly(propylene glycol)- and Pluronic (a PEG-PPG copolymer).

In some embodiments, the copolymer comprises about 10% of the firstpolymer monomer and about 90% of the second polymer monomer. In someembodiments, the copolymer comprises about 20% of the first polymermonomer and about 80% of the second polymer monomer. In someembodiments, the copolymer comprises about 30% of the first polymermonomer and about 70% of the second polymer monomer. In someembodiments, the copolymer comprises about 40% of the first polymermonomer and about 60% of the second polymer monomer. In someembodiments, the copolymer comprises about 50% of the first polymermonomer and about 50% of the second polymer monomer. In someembodiments, the first, second, and co-polymers phases are aqueous. Insome embodiments, the first, second, and co-polymers phases are organic.

2. Method of Analyzing an Analyte of Interest Based on Affinity

In yet another aspect, a novel MPS is used for analyzing or separatingan analyte of interest. In some embodiments, the separation of theanalyte is based on affinity. The analyte has preferential affinity forone phase over other phases of the MPS. In order for the MPS to be usedfor affinity analysis, the different phase components interact with theanalyte and preferentially partition into the phase with which it hasthe highest affinity.

In order to preferentially partition into one phase or another, theanalyte is subjected to simultaneous contact with all of the phases inthe novel multi-phase systems as described herein. The analyte and theMPS mixture is subjected to thorough mixing so that the analyte caninteract with the entire system and preferentially partitions into onephase as the mixed system phase separates. In some embodiments, theanalyte and the MPS mixture is stacked in a shaker or by hand. Othermethods of mixing known in the art are contemplated. Mixing creates anon-equilibrium state. The system may regain equilibrium by centrifugingor waiting for gravity to settle out the phases again.

Partitioning, as described in this manner, is well suited for smallanalytes. Exemplary small analytes include small molecules, proteins andpeptides, antibodies, oligonucleotides, organelles, viruses, and cells.Some analytes are small enough so that they are not suitable for densitybased separation. In these cases, the analytes can be subjected topartitioning in MPS, or affinity based separation using a MPS.

3. Method of Analyzing or Separating an Analyte of Interest Based onDensity

The multi-phase systems are well suited for analysis of analytes basedon density. That is, the MPS can distinguish among analytes based ondifferences in their density. In order to separate analytes in this way,the MPS system should have a density range that is close to that of theanalyst of interest. Of course it is possible that some of the analytescan be of higher density or lower density than the density range of theMPS, in which case they would occupy positions above and below the MPSrespectively during the separation process.

In some embodiments, the MPS is used to analyze an analyte of interest.Each phase of the MPS has an upper and a lower phase boundary, and themulti-phase system further comprises an analyte located at one of saidboundaries. In other words, the analyte may remain at the interface oftwo adjacent phases. This may be due to the fact that the density of theanalyte is between the two adjacent phases contacting the interface. Inother embodiments, the analyte may have the same density as that of oneof the phases and the analyte will remain in the phase withoutcontacting any boundary and between the upper and lower boundaries ofthat phase. In still other embodiments, the analyte may have a densityless than that of the top phase of the MPS (the phase with the leastdensity) and remain at the top of the MPS with a portion of the analyteabove the upper boundary of the top phase. In still other embodiments,the analyte may have a density more than that of the bottom phase of theMPS (the phase with the most density) and remain at the bottom of theMPS. Non-limiting examples of the analytes include solid particles,aggregates of small particles, plastic resins, wood fragments, glasses,and minerals.

In some specific embodiments, the analyte is subjected to sequentialcontact with one or more of the phases in the multi-phase system. Inthese embodiments, the analyte is separated based on its density. Theanalyte is introduced into a phase separated multi-phase system withoutdisturbing the phases and allowed to migrate to a location in themultiphase system that is characteristic of its density. Thisdensity-based separation is described in more details below.

In yet another aspect, a method of analyzing or separating a samplecomprising one or more analytes of interest using a multi-phase systemis described, comprising:

a) providing a multi-phase system comprising two or more phasesincluding at least two adjacent phases, wherein

-   -   each of the two adjacent phases comprises a phase component        selected from the group consisting of a polymer, a surfactant,        and a combination thereof;    -   at least one of the two adjacent phases comprises a polymer;    -   each of the two or more phases has a different density and the        two or more phases, taken together, represent a density        gradient; and    -   the two adjacent phases are phase-separated from each other;

b) introducing a sample comprising one or more analytes of interest tothe multi-phase system; and

c) allowing each of the analytes to migrate to a location in themultiphase system that is characteristic of its density, wherein duringmigration the sample contacts one or more of the two or more phasessequentially.

In some embodiments, each of the adjacent phases share a common solvent.In some embodiments, each of the adjacent phases share a common organicsolvent. The organic solvent can be selected from the groups consistingof liquid polymer, non-polar organic solvent, polar aprotic or proticsolvent, supercritical fluid, fuel, oils, and fluorinated solvents, andcombinations thereof. In some embodiments, each of the two or morephases comprises a phase component. In some embodiments, each of the twoor more phases is organic. In some embodiments, each of the two or morephases is aqueous. In some embodiments, the multi-phase system comprisesat least one aqueous phase and at least one organic phase. In somespecific embodiments, the MPS comprises a mixture of aqueous and organicphases. In some embodiments, the polymer is selected from the groupconsisting of homopolymer, block copolymer, copolymer, terpolymer,random copolymer, and a combination thereof. In some embodiments, thepolymer has a morphology selected from a group consisting of linearpolymer, branched polymer, crosslinked polymer, and dendrimer system.

In some specific embodiments, the multi-phase system further comprisesone or more additional phases selected from the group consisting ofsilicone oil, ionic liquid, fluorinated liquid, organic solvent, liquidmetal, and a combination thereof. The additional phases are not requiredto have a phase component.

In these embodiments, the analyte is introduced into the MPS and allowedto migrate through the one or more phases of the MPS, one phase at atime. Thus, the analyte will contact the phase(s) of the MPSsequentially and migrate to a location of MPS corresponding to itsdensity. In this process, the analyte does not have simultaneous contactwith two or more phases of the MPS except when passing the interfacebetween two adjacent phases. This method is distinguished from theseparation based on affinity as described above in that in the latter,the analyte needs to have simultaneous contact with all of the phases ofthe MPS so that a thermodynamic equilibrium is reached and the analytecan preferentially reside in one of the phases based on its affinitytowards that phase. This process is commonly referred to as‘partitioning’ or ‘extraction.’ In comparison, in the density-basedseparation as described herein, the analyte migrates through the MPSphase one at a time, contacting one or more of the phases sequentiallyand eventually arriving at a location in the MPS characteristic of itsdensity. Because the analyte only experiences a single phase at a time,no partitioning or extraction of the analyte into a particular phase ispossible.

Samples comprising the analyte(s) can be introduced to the MPS in theform of a solution or suspension of material. Non-limiting examples ofways in which these samples can be added to the MPS include by pour,pipette, injection, drip, siphon, capillary action, spray, aspirationfollowed by expulsion, and pump.

In some embodiments, the multi-phases is provided as dispersion oremulsion in another carrier phase. The multiphase system can be providedin the form of droplets, and they can be dispersed in an oil orfluorinated solvent to be collected/combined prior to use.

Each phase of the MPS has an upper and a lower phase boundary, and twoadjacent phases forms a common interface in between. In most instances,there is not an exact match between the analyte density and the densityof any particular phase. The analyte's density is between the densitiesof two adjacent phases in a MPS, and the analyte should therefore remainat the interface of the two adjacent phases. If the analyte should havethe same density as that of one of the phases, the analyte will remainwith in the density-matched phase without contacting any boundary. Inthis case, the analyte resides within the phase due to a density matchand not due to any favorable or preferential interaction of the analytewith one phase over another. In still other embodiments, the analyte mayhave a density less than that of the top phase of the MPS (the phasewith the least density) and remain at the top of the MPS with a portionof the analyte above the upper boundary of the top phase aftermigration. In still other embodiments, the analyte may have a densitymore than that of the bottom phase of the MPS (the phase with the mostdensity) and remain at the bottom of the MPS after migration.

A density-based separation of a sample containing one or more analytesusing MPS is described with reference to FIG. 6. As shown in FIG. 6, atwo-phase MPS (a dextran-Ficoll ATPS) is used for separation of threebeads with different densities. A two-phase MPS functions as a two-stepsink/float assay and provides three possible equilibrium positions foran object based on the differences in density between the object and itsenvironment: (i) at the interface between air and the top phase, (ii) atthe interface between the top and bottom phases, and (iii) at theinterface between the bottom phase and the container. As shown in FIG.6, the first bead, with a density less than the density of the topphase, will reside at the upper boundary of the top phase. The secondbead, with a density less than the density of the bottom phase buthigher than the top phase, will reside at the interface between the topand the bottom phases. Lastly, the third bead, with a density higherthan the densities of the top and bottom phases, will reside at bottomof the MPS and at the interface between the bottom phase and thecontainer. In some embodiments, using the density step produced by theMPS, the objects with density difference of less than 0.100 g/cm³ can beseparated. In some embodiments, using the density step produced by theMPS, the objects with density difference of less than 0.050 g/cm³ can beseparated. In some embodiments, using the density step produced by theMPS, the objects with density difference of less than 0.010 g/cm³ can beseparated. In some embodiments, using the density step produced by theMPS, the objects with density difference of less than 0.005 g/cm³ can beseparated. In some embodiments, using the density step produced by theMPS, the objects with density difference of less than 0.004 g/cm³ can beseparated. In some embodiments, using the density step produced by theMPS, the objects with density difference of less than 0.002 g/cm³ can beseparated. In some embodiments, using the density step produced by theMPS, the objects with density difference of less than 0.001 g/cm³ can beseparated.

In some embodiments, the analyte is allowed to migrate based on gravity.In other embodiments, the analyte is allowed to migrate using acentrifuge. Non-limiting examples of centrifuge include laboratorycentrifuges (using either a fixed angle or swinging bucket rotors) or asoft-centrifuge. Soft centrifugation refers the uses of soft tubing,e.g., polyethylene tubing, as the sample container and a simple deviceas the rotor (see, Wong et al., “Egg beater as centrifuge: isolatinghuman blood plasma from whole blood in resource-poor setting”, Lab Chip,2008, 8, 2032-2037). In some specific embodiments, the softcentrifugation is achieved by an eggbeater centrifuge. Other methods ofsoft centrifugation known in the art are also contemplated.

Migration occurs until the analyte reaches a phase with which it isdensity matched or a phase of higher density, so that it no longer movesover time through the multi-phase system. This situation is referred toas having reached an ‘equilibrium location’. In the case of gravitymigration, the time to reach equilibrium migration is in the range ofseconds, minutes, hours, days, or more. This depends on the viscosity ofthe phases, density difference between the phase and the analyte, andthe size/shape of the analyte. Use of centrifugation can accelerate themigration process and reduce the time to reach equilibrium location. Thecentrifuge works using the sedimentation principle, where thecentripetal acceleration causes more dense substances to separate outalong the radial direction (the bottom of the tube). By the same token,lighter objects will tend to move to the top. The centrifugation can berun at different temperatures such as 4° C., 22° C., 37° C., or 60° C.Additionally, the centrifugation can be run at from low speed (severalmultiples of g) to 170,000 g or more. The centrifugation can be run fromseconds, minutes, hours, or days. The centrifugation can be run usingswinging bucket or fixed angle.

Because the separation is carried out using gravity (enhancedgravitational force using centrifugation), the analyte is desirably insuspension in the MPS phases, e.g., the analyte is insoluble in the MPSphases. In addition, separation will be achieved more readily and in ashorter time frame for larger analytes. Without additionalmodifications, such as aggregating smaller particles into largeraggregates or tagging smaller particles to increase size and/or density,and objects having a dimension of greater than 100 nm, greater than 200nm, greater than 500 nm, greater than 750 nm or greater than 1 m can beseparated. There is no upper limit to the size of the particles that canbe separated using the MPS; however, practical considerations such asthe size of the density column may limit its application.

In embodiments in which the sample comprises small particles that are ofinterest, the samples can be subjected to aggregating agents to induceaggregation of the small particles, so that the analyte is larger andcan be separated readily using MPS. or the densities of the smallparticles can be modified using additives to force their migration todifferent locations in the MPS such that the aggregated small particlespass through one or more phases sequentially. If small particles, suchas viruses or cells, have multiple copies of a ligand on their surface,they can be aggregated using reagents functionalized with multiplecopies of molecules that recognize that ligand. Non-limiting examples ofaggregating agents include multivalent particles (for viruses, cells,anything expressing a surface marker), adenosine diphosphate (forplatelets), hemagglutinin (for erythrocytes), concanavalin A (forerythrocytes). For example, a CD4+ T cell and a microparticlefunctionalized with an antibody to CD4; adenosine diphosphate aggregatesplatelets; hemagglutinin aggregates erythrocytes; and DNA aggregatesgold colloids. The ranges of sizes for the aggregates can be from 10 nmto 100 μm.

Various additives can be added to the phases of MPS. Non-limitingexamples of additives include salt, D₂O, buffered water, and polymersand surfactant whose amounts are not sufficient to affect a phaseseparation.

As used herein, the density range of a MPS refers to the range from thelowest density of the phases in MPS, i.e., the density of the top phase,to the highest density of the phases in the MPS, i.e., the density ofthe bottom phase. The range of the densities of the phases in MPS can beshifted or modified, e.g., broadened. A MPS has a density range set bythe top phase (lowest density) and the bottom phase (highest density).Accordingly, if two or more analytes all have densities lower than thedensity of the top phase in a MPS, the two or more analyte will allreside at the upper boundary of the top phase in a density-basedseparation and remain unseperated. Likewise, if two or more analytes allhave densities higher than the density of the bottom phase in a MPS, thetwo or more analyte will all reside at the lower boundary of the bottomphase, i.e., the bottom of the MPS, in a density-based separation andremain unseparated.

In some embodiments, the lower end of the density range of an existingMPS can be broadened by adding additional phase(s) with densities lowerthan the top phase of the existing MPS. The additional phases can phaseseparate from the phases of the existing MPS and form a new MPS. Thus,the density range of the MPS is effectively broadened by lowering thelower end of the density range. The additional phases can be one or moreaqueous phases comprising phase components, or a pure organic or aqueoussolvent.

In some embodiments, the upper end of the density range of an existingMPS can be broadened by adding additional phase(s) with densities higherthan the bottom phase of the existing MPS. The additional phases canphase separate from the phases of the existing MPS and form a new MPS.Thus, the density range of the MPS is effectively broadened byincreasing the upper end of the density range. The additional phases canbe one or more aqueous phases comprising phase components, or a pureorganic or aqueous solvent.

In some embodiments, each phase of the MPS may have a common solvent. Insome embodiments, the density range of a MPS having a common solvent canbe shifted by adding one or more additives to the phases of the MPS. Insome embodiments, the additive may be soluble in or mixable with thecommon solvent and thus evenly distributed in each phase of the MPS.Non-limiting examples of the additives include co-solvent, salt, and acombination thereof. The additive may have a density higher or lowerthan the common solvent. As a result, the density of each phase may allincrease or decrease, respectively, upon the addition of the additive.

In some embodiments, the density range of the MPS can be shifted byadding a co-solvent. The co-solvent may be miscible with the commonsolvent of the MPS and have a density higher or lower than the commonsolvent of the MPS. In some embodiments, the co-solvent is evenlydistributed in all of the MPS phases. In these instances, if aco-solvent with a density higher than the common solvent is added toeach phase of the MPS, the density of each phase of the MPS willincrease. Alternatively, if a co-solvent with a density lower than thecommon solvent is added to each phase of the MPS, the density of eachphase of the MPS will decrease. In either case, the density range of theoriginal MPS will be shifted. In some specific embodiments, the commonsolvent of MPS is water and the co-solvent is D₂O. D₂O has a densityhigher than water and thus the density of a water phase will be lowerthan that of a phase containing water and D₂O as co-solvent (under thesame phase component concentrations). In this case, each phase's densitywill increase.

In some embodiments, the density range of the MPS can be shifted byadding salt. The salt can dissolve in the common solvent of the MPS andis evenly distributed in all of the MPS phases. The salt may have adensity higher than the common solvent of the MPS. Non-limiting examplesof salts include alkali metal or alkali earth metal halide, phosphate,sulfate, carbonate. Other salts commonly known in the art arecontemplated. In some specific examples, the salt is selected from thegroup consisting of LiBr, NaBr, KBr, RbBr, CsBr, and a combinationthereof. The density of the salt is usually higher than the commonsolvent of the MPS, therefore the density of each phase of the MPS willincrease. Alternatively, other small molecules with low densities andsoluble in aqueous or organic solvents can be used as additives todecrease the density of a phase.

As a result of the disclosed methods, one or more analytes of interestmay preferentially accumulate in one of the phase or at an interface inthe MPS, while another analyte, impurity or object in the samplecontaining the analyte may preferentially accumulate in another phase orinterface of the MPS. The desired analyte in the sample can bevisualized after separation via a variety of methods. Firstly,separation of some analytes can visualized by human eye. Those that arenot readily visible by the eye can be visualized using methods known inthe art. For example, separation can be visualized with the aid of amicroscope and magnifying glass or by using fluorescent dyes.

In some embodiments, it will be sufficient to simply observe thelocation of the analyte in the multi-phase system. Suitable analytes forobservation by eyes include beads, cells, plastic resins, plastics,glitter, minerals, gems, archaeological species (bone and dirt andclay), etc.

In other embodiments, the desired analyte in the sample can be recoveredby retrieving the phase that this analyte preferentially has accumulatedin, thus resulting in an improved purity of such analyte. Analytes canbe recovered from the system using extraction methods known in the art.In several aspects of one or more embodiments, analytes retained ingradients can be recovered using a fractionator, pipette, drip method,side-puncturing a tube, or combinations thereof. In one aspect, afractionator can be used to carefully control the pressure on the liquidand pull known volumes of the gradient in certain increments. The dripmethod can also be used to extract separated analytes. The bottom of atube is punctured and allowed to drip into sample tubes. This method,like the fractionator method, is ideal for systems such as the disclosedMPS that form clear visual interfaces that can be observed by eye. Inanother aspect, a pipette is introduced to the top of the sample toremove most, but not all, of the top layer without pulling too close tothe interface. Once the top layer is mostly removed, a clean pipette tipcan be inserted from the top layer into the second layer. Lightagitation of the tip can be used to clear the interface from the tip.The desired layer can then be drawn up in the pipette. The interfacesabove and below the desired layer should not be drawn up with thedesired layer to avoid layer contamination. In yet another aspect, aplastic tube is side punctured one or more times using a needle, such asa 21 to 16 gauge needle, to puncture the tube at the desired phase. Thedesired phase is pulled from the tube volume. In each of these aspects,if the analyte of interest is in a phase, the interfaces above and belowthe phase should not be disturbed to avoid layer contamination.Similarly, if the analyte of interest is in an interface, the phasesabove and below the interface should not be disturbed to avoid layercontamination.

The sample containing the analyte can be liquid, e.g., liquid droplets(immiscible in that various phases of the MPS), a solid, gel, or liquidcrystal. The sample is selected from the group consisting of forensicsstudy sample, a sample indicative of animal health, a sample indicativeof human identity used for border control, home land security, orintelligence, a sample from food processing, a sample indicative ofproduct quality, a sample indicative of environmental safety, a samplecontaining different crystal polymorphs, and combinations thereof. Byway of example, rocks and bones can be separated by density. This mayassist in forensic investigations or archaeology where it is desired toremove impurities found with valuable evidence. It also may be able todistinguish between different fragments of materials. Because the methodis non-destructive, it can be used to separate a materials and theforensic evidence can be further analyzed. Other non-limiting examplesinclude crystallites, mineral, biomineral (bone), composite, plastic,textile, wood, and all applications of those species. For instance, theMPS described herein can be used for checking for viable seeds, orgrains, detecting oils of plant origins, or distinguishing differenttype of food products including cheeses, peanut butter, and honeys.

In some embodiments, a two-phase MPS can be used for purposes disclosedherein. In some other embodiments, three or more phase systems are used.It was believed that the inclusion of additional phases may prevent theenrichment of the target molecule in a specific phase, because thetarget molecule may distribute into the additional phases. This beliefmay account for the lack of literature regarding these multi-phasepolymer systems. Applicants have surprisingly found that broadening thelandscape of polymers that demonstrate immiscibility in aqueousmulti-phase polymer systems provides superior tunability forapplications based on differences in density and affinity and finercontrol over the partitioning of complex mixtures of subjects.

In some embodiments, the multi-phase polymer system is provided bymixing suitable polymers or surfactants with a solvent and subjectingthe mixture to centrifugation. For instance, a mixture of solid polymersor surfactants can be mixed with a common solvent, e.g., water, to allowthe resulting mixture to phase-separate to form a MPS. Any types ofcentrifugation known in the art can be used in the formation of the MPS.In some embodiments, the MPS is formed using soft centrifugation. Softcentrifugation is described above. In some specific embodiments, thesoft centrifugation is achieved by an eggbeater centrifuge. Othermethods of soft centrifugation known in the art are also contemplated.

The method Applicants report here combines the portability andsimplicity of the soft centrifuge with aqueous multiphase densitybarriers generated from immiscible polymers or surfactants. Immisciblepolymers or surfactants have numerous advantages over discontinuousdensity gradients for field use: they are easily prepared, owing to thenature of their mutual immiscibility; they are stable, and thus amenableto long term storage; and they are versatile, as Applicants havepreviously identified a suite of multiphase systems that can be altered(composition and/or density) to suit the application.

3. Method of Determining an Object's Density Using a MPS

All matter is characterized by physical properties (e.g., mass,conductivity, and permittivity). As measurable quantities, the use ofphysical properties to compare objects has the potential to be widelyapplicable to a range of problems. Differences in density, for example,have been used to analyze the composition or purity of samples andmonitor chemical processes. One approach to density analysis is thesink/float assay—an object is introduced to a solution and is eithermore dense (i.e., it sinks) or less dense (i.e., it floats) than thesolution. This assay, and its binary method of separation, hasapplications in recovering archaeological samples from soil, liberatingmetals from plastics in scrap recycling, and isolating biologicalmaterials. Using this approach, applications requiring the density-basedseparation of multiple components involve a series of solutions with arange of densities. In some embodiments, a MPS produced from a mixtureof immiscible liquids is used to determine the density of an object.Mixtures of liquids that result in phase separation include oil/water,dichloromethane/water, aqueous two-phase polymer systems (ATPS), and MPSas described herein.

In yet another aspect, a method of determining the density of a solidparticle using a multi-phase system is described, comprising:

a) providing a multi-phase system comprising two or more phases, wherein

-   -   at least one of the phases comprises a phase component, wherein        the phase component is selected from the group consisting of a        polymer, a surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        two or more phases, taken together, represent a density        gradient; and    -   the phases are phase-separated from each other;

b) introducing a solid particle to the multi-phase system;

c) allowing the solid particle to migrate to a location in themultiphase system that is characteristic of its density, wherein duringmigration the sample contacts one or more of the two or more phasessequentially; and

d) determining the density of the solid particle.

In some embodiments, the solid particle is within one of the phasesafter migration and the density of the solid particle is measured as thedensity of the phase the containing the solid particle.

In some embodiments, the solid particle is a bead residing in aninterface between two adjacent phases and the density of the bead iscalculated based on the position of the bead, the buoyancy, and theinterfacial tension between the two phases. In these embodiments, thebead is captured at the liquid/liquid interface of a MPS, and adisplacement of interface can be observed (see FIG. 7) proportional todifferential buoyant density of the bead across the density step. Bybalancing the gravitational, buoyant, and interfacial forces that act onthe bead at the liquid/liquid interface, an equation is derived capableof calculating the density of the bead to within an accuracy of 1% usingmeasured geometric parameters (e.g., bead radius and contact angle).This equation is used to calculate the unknown density of a polystyrenebead, e.g., a polystyrene bead.

Current approaches to density-based separations employ gradients: (i)kinetic density gradients must be produced in situ by centrifugationduring a separation, centrifugation parameters (e.g., time and relativecentrifugal force) require optimization, and do not provide interfacesfor sample collection; and (ii) sequential step gradients (e.g., usingincreasing concentrations of sucrose or Ficoll) require skill toprepare, must be prepared immediately prior to use, and the interfacesbetween layers are under thermodynamic control (e.g., diffusion andmixing will destroy the interface). In contrast, density steps preparedby phase separation offer many ideal features: (i) steps formspontaneously after mixing, (ii) the systems are at equilibrium and maybe prepared in advance, (iii) the magnitude of the density step can beadjusted by changing the concentrations of the components that compriseeach phase, and (iv) the interface between phases is well-defined andthus useful for isolating objects after separation.

In some embodiments, beads of known density are introduced to an ATPSmanually and allowed to settle under gravity (i.e., Ig). The differencesin density between the bead and each layer of the ATPS (i.e., thebuoyant density) controls the rates by which the bead migrates throughthe system. For example, the beads required approximately ten minutes tomigrate completely through the small density step produced by the ATPS(for a dextran-Ficoll ATPS, the zp between the two phases is 0.0023g/cm³). In some embodiments, centrifugal force is applied to speed upthe migration. The applied centrifuge increases the sedimentation rateof each bead, but does not affect the final position of the bead.

Referring back to FIG. 6, three beads are separated by using the densitystep produced by the ATPS, e.g., a dextran-Ficoll ATPS, with one bead ateach possible interface (FIG. 6). In some embodiments, the differencesin density between the beads in this set can be very small, e.g., lessthan about 0.0020 g/cm³, but the density step between phases in the ATPSresolves each bead readily. Separations of beads with larger differencesin density using a larger density step are also possible (e.g.,PEG-Ficoll ATPS; FIG. 8).

The position of the bead in the interface can be used to measuredensity. While the layer/layer interface captured beads predictablybased on density, the presence of the bead displaced partially theotherwise continuous interface of the ATPS (FIG. 7A). As a result, theabsolute position of the bead varied in a manner that depended on (i)the density of the bead, (ii) the density of each layer, and (iii) thecontact angle at the bead/liquid/liquid interface. There are threeforces acting on a bead residing at the interface of an ATPS: (i)gravity (F_(g); N), (ii) buoyancy (F_(B); N), (iii) and the interfacialtension (F_(I); N). At equilibrium, these forces are balanced (equation1).{hacek over (F)} _(g) ={hacek over (F)} _(B) +{hacek over (F)} _(I)  (1)

In some embodiments, the density of the solid object or particle can becalculated based on equation 1. In some specific embodiments, the solidobject is a bead and the density of the bead (ρ₀; g/cm³) can becalculated as a function of the density of the top phase (ρt; g/cm³),the density of the bottom phase (ρ_(b); g/cm³), the acceleration due togravity (g; m/s²), the interfacial tension between the top and bottomlayers (γ_(bt); N/m), the surface/liquid/liquid contact angle (θ_(c);deg), the angle between the bead center and the bead/interface point ofintersection (φ; deg), the bead radius (R; m), and the displacement ofthe interface (d; m) (equation 2; for a full derivation, please seeexperimental section). Equation 2 comprises terms for the mean solutiondensity, a correction for buoyancy, and a correction for surfacetension.

$\begin{matrix}{\rho_{0} = {\frac{\left( {\rho_{b} + \rho_{t}} \right)}{2} + {\frac{\left( {\rho_{b} - \rho_{t}} \right)}{4}\left\lfloor {{\cos^{3}\phi} - {3\cos\;\phi} + {3\frac{d}{R}\sin^{2}\phi}} \right\rfloor} + {\frac{3\gamma_{bt}}{2{gR}^{2}}\sin\;{{\phi cos}\left( {\theta_{c} + \phi - \pi} \right)}}}} & (2)\end{matrix}$

In some embodiments, the density of a bead of unknown density iscalculated geometrically using equation (2) from several measuredvariables (FIG. 7B).

Preparation of Specific Multiphase Systems

In one aspect, a multi-phase system comprising two or more phases isdescribed, wherein

-   -   each phase comprises a phase component selected from the group        consisting of a polymer, a surfactant, and a combination        thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(vinyl alcohol), poly(2-ethyl-2-oxazoline), poly(ethylene        glycol), dextran, Ficoll, and Tween;        with the proviso that the multi-phase system does not include:

an aqueous three-phase system wherein the phase components in the threephases are a combination of poly(ethylene glycol)-dextran-Ficoll; or

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofpoly(vinyl alcohol)-poly(ethylene glycol), poly(vinyl alcohol)-dextran,dextran-Ficoll, poly(ethylene glycol)-Ficoll, poly(ethyleneglycol)-dextran, poly(ethylene glycol)-Tween, and dextran-Tween. Thesolvent for each phase can be aqueous or organic. In some embodiments,the MPS comprises a mixture of aqueous and organic phases.

In some embodiments, the multi-phase system is a six-phase system andthe phase components in the six phases are poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), dextran, Ficoll, andTween, respectively. In some specific embodiments, each phase of the MPSis aqueous phase.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are a combination of phasecomponents selected from the group consisting of

poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethyleneglycol)-dextran-Ficoll,

poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethyleneglycol)-dextran-Tween,

poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethyleneglycol)-Ficoll-Tween,

poly(vinyl alcohol)-poly(ethylene glycol)-dextran-Ficoll-Tween,

poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-dextran-Ficoll-Tween, and

poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran-Ficoll-Tween. Insome specific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-dextran,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Ficoll,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Tween,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-dextran-Ficoll,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-dextran-Tween,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Ficoll-Tween,-   poly(vinyl alcohol)-poly(ethylene glycol)-dextran-Ficoll,-   poly(vinyl alcohol)-poly(ethylene glycol)-dextran-Tween,-   poly(vinyl alcohol)-poly(ethylene glycol)-Ficoll-Tween,-   poly(vinyl alcohol)-dextran-Ficoll-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Ficoll-Tween,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll-Tween, and-   poly(ethylene glycol)-dextran-Ficoll-Tween. In some specific    embodiments, each phase of the four-phase MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-dextran,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Tween,-   poly(vinyl alcohol)-poly(ethylene glycol)-dextran,-   poly(vinyl alcohol)-poly(ethylene glycol)-Ficoll,-   poly(vinyl alcohol)-poly(ethylene glycol)-Tween,-   poly(vinyl alcohol)-dextran-Ficoll,-   poly(vinyl alcohol)-dextran-Tween,-   poly(vinyl alcohol)-Ficoll-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Ficoll,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Tween,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-dextran-Tween,-   poly(2-ethyl-2-oxazoline)-Ficoll-Tween,-   poly(ethylene glycol)-dextran-Tween,-   poly(ethylene glycol)-Ficoll-Tween, and-   dextran-Ficoll-Tween. In some specific embodiments, each phase of    the three-phase MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline),-   poly(ethylene glycol)-poly(2-ethyl-2-oxazoline),-   poly(ethylene glycol)-dextran,-   poly(vinyl alcohol)-Ficoll,-   poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(vinyl alcohol)-Tween,-   poly(2-ethyl-2-oxazoline)-Tween, and-   Tween 20-Ficoll. In some specific embodiments, each phase of the    two-phase MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), poly(2-ethyl-2-oxazoline), poly(ethylene glycol), polyacrylamide,polyethyleneimine, and CHAPS. The solvent for each phase can be aqueousor organic. In some embodiments, the MPS comprises a mixture of aqueousand organic phases. In some embodiments, the MPS comprises all aqueousphases. In some embodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a six-phase system andthe phase components in the six phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), polyacrylamide,polyethyleneimine, and CHAPS, respectively. In some specificembodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide-CHAPS,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-polyacrylamide-CHAPS,-   poly(methacrylic acid)-poly(ethylene    glycol)-polyethyleneimine-polyacrylamide-CHAPS, and-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine-polyacrylamide-CHAPS. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide-polyethyleneimine,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-polyethyleneimine-poly(ethylene    glycol)-polyacrylamide,-   poly(methacrylic acid)-poly(ethylene glycol)-polyacrylamide-CHAPS,-   poly(methacrylic acid)-poly(ethylene    glycol)-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-polyacrylamide-polyethyleneimine-CHAP S,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine-CHAPS,-   poly(2-ethyl-2-oxazoline)-polyacrylamide-polyethyleneimine-CHAPS,    and-   poly(ethylene glycol)-polyacrylamide-polyethyleneimine-CHAPS. In    some specific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol),-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-CHAPS,-   poly(methacrylic acid)-poly(ethylene glycol)-polyacrylamide,-   poly(methacrylic acid)-poly(ethylene glycol)-polyethyleneimine,-   poly(methacrylic acid)-poly(ethylene glycol)-CHAPS,-   poly(methacrylic acid)-polyacrylamide-polyethyleneimine,-   poly(methacrylic acid)-polyacrylamide-CHAPS,-   poly(methacrylic acid)-polyethyleneimine-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly acrylamide-poly ethyleneimine,-   poly(2-ethyl-2-oxazoline)-polyacrylamide-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly ethyleneimine-CHAPS,-   poly(ethylene glycol)-polyacrylamide-polyethyleneimine,-   poly(ethylene glycol)-polyacrylamide-CHAPS,-   poly(ethylene glycol)-polyethyleneimine-CHAPS, and-   polyacrylamide-polyethyleneimine-CHAPS. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline),-   poly(methacrylic acid)-poly(ethylene glycol),-   poly(methacrylic acid)-polyacrylamide,-   poly(methacrylic acid)-polyethyleneimine,-   poly(methacrylic acid)-CHAPS,-   poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(2-ethyl-2-oxazoline)-CHAPS,-   poly(ethylene glycol)-polyethyleneimine,-   poly ethyleneimine-poly acrylamide,-   polyethyleneimine-CHAPS,-   polyacrylamide-poly(ethylene glycol),-   poly(ethylene glycol)-CHAPS, and-   polyacrylamide-CHAPS. In some specific embodiments, each phase of    the two-phase MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(methacrylic acid), poly(2-ethyl-2-oxazoline),        polyacrylamide, polyethyleneimine, Pluronic and CHAPS. Pluronic        F68 is the species of PEG-PPG co-polymer that is a member of a        large family of PEG-PPG block copolymers. It is contemplates        that others in the family will have similar properties.

In some embodiments, the multi-phase system is a six-phase system andthe phase components in the six phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), polyacrylamide, polyethyleneimine, Pluronic,and CHAPS, respectively. In some embodiments, the MPS comprises amixture of aqueous and organic phases.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide-polyethyleneimine-Pluronic,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide-Pluronic-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-Pluronic-CHAPS,-   poly(methacrylic    acid)-polyacrylamide-polyethyleneimine-Pluronic-CHAPS, and-   poly(2-ethyl-2-oxazoline)-polyacrylamide-polyethyleneimine-Pluronic-CHAPS.    In some specific embodiments, each phase of the MPS is aqueous    phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide-Pluronic,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-Pluronic,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Pluronic-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide-polyethyleneimine,-   poly(methacrylic acid)-polyacrylamide-polyethyleneimine-Pluronic,-   poly(methacrylic acid)-polyacrylamide-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-polyacrylamide-Pluronic-CHAPS,-   poly(methacrylic acid)-polyethyleneimine-Pluronic-CHAPS,-   polyacrylamide-poly(2-ethyl-2-oxazoline)-polyethyleneimine-Pluronic    F68,-   polyacrylamide-poly(2-ethyl-2-oxazoline)-polyethyleneimine-CHAPS,-   polyacrylamide-poly(2-ethyl-2-oxazoline)-Pluronic-CHAPS,-   polyethyleneimine-poly(2-ethyl-2-oxazoline)-Pluronic-CHAPS, and-   polyacrylamide-polyethyleneimine-Pluronic-CHAPS. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Pluronic,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-CHAPS,-   poly(methacrylic acid)-polyacrylamide-polyethyleneimine,-   poly(methacrylic acid)-polyacrylamide-Pluronic,-   poly(methacrylic acid)-polyacrylamide-CHAPS,-   poly(methacrylic acid)-polyethyleneimine-Pluronic,-   poly(methacrylic acid)-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-Pluronic-CHAPS,-   poly(2-ethyl-2-oxazoline)-polyacrylamide-poly ethyleneimine,-   poly(2-ethyl-2-oxazoline)-polyacrylamide-Pluronic,-   poly(2-ethyl-2-oxazoline)-polyacrylamide-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly ethyleneimine-Pluronic,-   poly(2-ethyl-2-oxazoline)-poly ethyleneimine-CHAPS,-   poly(2-ethyl-2-oxazoline)-Pluronic-CHAPS,-   poly acrylamide-poly ethyleneimine-CHAPS,-   poly acrylamide-poly ethyleneimine-Pluronic,-   polyacrylamide-Pluronic-CHAPS, and-   polyethyleneimine-Pluronic-CHAPS. In some specific embodiments, each    phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline),-   poly(methacrylic acid)-Pluronic,-   poly(methacrylic acid)-polyacrylamide,-   poly(methacrylic acid)-polyethyleneimine,-   poly(methacrylic acid)-CHAPS,-   poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-Pluronic,-   poly(2-ethyl-2-oxazoline)-CHAPS,-   poly(ethylene glycol)-polyethyleneimine,-   poly ethyleneimine-poly acrylamide,-   polyethyleneimine-CHAPS,-   poly acrylamide-Pluronic,-   Pluronic-CHAPS, and-   polyacrylamide-CHAPS. In some specific embodiments, each phase of    the two-phase MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(methacrylic acid), poly(2-ethyl-2-oxazoline), poly(ethylene        glycol), polyethyleneimine, Ficoll, and CHAPS, with the proviso        that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of poly(ethylene glycol)-Ficoll. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.

In some embodiments, the multi-phase system is a six-phase system andthe phase components in the six phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), Ficoll,polyethyleneimine, and CHAPS, respectively. In some specificembodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Ficoll-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Ficoll-CHAPS, poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-Ficoll-CHAPS,-   poly(methacrylic acid)-poly(ethylene    glycol)-polyethyleneimine-Ficoll-CHAPS, and-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine-Ficoll-CHAPS. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly ethyleneimine-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-poly(methacrylic acid),-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Ficoll,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-Ficoll-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Ficoll-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-polyethyleneimine-poly(ethylene    glycol)-Ficoll,-   poly(methacrylic acid)-poly(ethylene glycol)-Ficoll-CHAPS,-   poly(methacrylic acid)-poly(ethylene    glycol)-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-Ficoll-polyethyleneimine-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Ficoll-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Ficoll-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine-CHAPS,-   poly(2-ethyl-2-oxazoline)-Ficoll-polyethyleneimine-CHAPS, and-   poly(ethylene glycol)-Ficoll-polyethyleneimine-CHAPS. In some    specific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol),-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-CHAPS,-   poly(methacrylic acid)-poly(ethylene glycol)-Ficoll,-   poly(methacrylic acid)-poly(ethylene glycol)-polyethyleneimine,-   poly(methacrylic acid)-poly(ethylene glycol)-CHAPS,-   poly(methacrylic acid)-Ficoll-polyethyleneimine,-   poly(methacrylic acid)-Ficoll-CHAPS,-   poly(methacrylic acid)-polyethyleneimine-CHAPS,-   polyethyleneimine-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Ficoll,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-CHAPS,-   poly(2-ethyl-2-oxazoline)-Ficoll-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-Ficoll-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly ethyleneimine-CHAPS,-   poly(ethylene glycol)-Ficoll-polyethyleneimine,-   poly(ethylene glycol)-Ficoll-CHAPS,-   poly(ethylene glycol)-polyethyleneimine-CHAPS, and-   Ficoll-polyethyleneimine-CHAPS. In some specific embodiments, each    phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline),-   poly(methacrylic acid)-poly(ethylene glycol),-   poly(methacrylic acid)-Ficoll,-   poly(methacrylic acid)-polyethyleneimine,-   poly(methacrylic acid)-CHAPS,-   poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(2-ethyl-2-oxazoline)-CHAPS,-   poly(ethylene glycol)-polyethyleneimine,-   polyethyleneimine-Ficoll,-   polyethyleneimine-CHAPS,-   poly(ethylene glycol)-CHAPS, and-   Ficoll-CHAPS. In some specific embodiments, each phase of the MPS is    aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(methacrylic acid), poly(2-ethyl-2-oxazoline),        polyethyleneimine, Pluronic F68, Ficoll, and CHAPS. In some        embodiments, the MPS comprises a mixture of aqueous and organic        phases.

In some embodiments, the multi-phase system is a six-phase system andthe phase components in the six phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), Pluronic F68, Ficoll, polyethyleneimine, andCHAPS, respectively. In some specific embodiments, each phase of the MPSis aqueous phase.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-Pluronic-Ficoll-polyethyleneimine,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-Pluronic-Ficoll-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-Pluronic-polyethyleneimine-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-Ficoll-CHAPS,-   poly(methacrylic acid)-Pluronic-polyethyleneimine-Ficoll-CHAPS, and-   poly(2-ethyl-2-oxazoline)-Pluronic-polyethyleneimine-Ficoll-CHAPS.    In some specific embodiments, each phase of the MPS is aqueous    phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Pluronic-Ficoll,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Pluronic-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-Ficoll-polyethyleneimine,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-Pluronic-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Ficoll-CHAPS,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-polyethyleneimine-Pluronic-Ficoll,-   poly(methacrylic acid)-Pluronic-Ficoll-CHAPS,-   poly(methacrylic acid)-Pluronic-polyethyleneimine-CHAPS,-   poly(methacrylic acid)-Ficoll-polyethyleneimine-CHAPS,-   poly(2-ethyl-2-oxazoline)-Pluronic-Ficoll-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-Pluronic-Ficoll-CHAPS,-   poly(2-ethyl-2-oxazoline)-Pluronic-poly ethyleneimine-CHAPS,-   poly(2-ethyl-2-oxazoline)-Ficoll-polyethyleneimine-CHAPS, and-   Pluronic F68-Ficoll-polyethyleneimine-CHAPS. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Pluronic,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-CHAPS,-   poly(methacrylic acid)-Pluronic-Ficoll,-   poly(methacrylic acid)-Pluronic-polyethyleneimine,-   poly(methacrylic acid)-Pluronic-CHAPS,-   poly(methacrylic acid)-Ficoll-polyethyleneimine,-   poly(methacrylic acid)-Ficoll-CHAPS,-   poly(methacrylic acid)-polyethyleneimine-CHAPS,-   poly(2-ethyl-2-oxazoline)-Pluronic-Ficoll,-   poly(2-ethyl-2-oxazoline)-Pluronic-CHAPS,-   poly(2-ethyl-2-oxazoline)-Pluronic-poly ethyleneimine,-   poly(2-ethyl-2-oxazoline)-Ficoll-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-Ficoll-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly ethyleneimine-CHAPS,-   Pluronic-Ficoll-poly ethyleneimine,-   Pluronic-Ficoll-CHAPS,-   Pluronic-polyethyleneimine-CHAPS, and-   Ficoll-polyethyleneimine-CHAPS. In some specific embodiments, each    phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline),-   poly(methacrylic acid)-Pluronic,-   poly(methacrylic acid)-Ficoll,-   poly(methacrylic acid)-polyethyleneimine,-   poly(methacrylic acid)-CHAPS,-   poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(2-ethyl-2-oxazoline)-Pluronic,-   poly(2-ethyl-2-oxazoline)-CHAPS,-   Pluronic F68-polyethyleneimine,-   polyethyleneimine-Ficoll,-   polyethyleneimine-CHAPS,-   Pluronic-Ficoll,-   Pluronic-CHAPS, and-   Ficoll-CHAPS. In some embodiments, the MPS comprises a mixture of    aqueous and organic phases.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), poly(2-ethyl-2-oxazoline), poly(ethylene glycol), Ficoll, andTween, with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consistingpoly(ethylene glycol)-Ficoll, and poly(ethylene glycol)-Tween. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), Ficoll, and Tween,respectively. In some specific embodiments, each phase of the MPS isaqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Ficoll,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Ficoll-Tween,-   poly(methacrylic acid)-poly(ethylene glycol)-Ficoll-Tween, and-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Ficoll-Tween. In    some specific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, wherein the multi-phase system is a three-phasesystem and the phase components in the three phases are a combination ofphase components selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol),-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Tween,-   poly(methacrylic acid)-poly(ethylene glycol)-Ficoll,-   poly(methacrylic acid)-poly(ethylene glycol)-Tween,-   poly(methacrylic acid)-Ficoll-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Ficoll,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Tween,-   poly(2-ethyl-2-oxazoline)-Ficoll-Tween, and-   poly(ethylene glycol)-Ficoll-Tween. In some specific embodiments,    each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline),-   poly(methacrylic acid)-poly(ethylene glycol),-   poly(methacrylic acid)-Tween,-   poly(methacrylic acid)-Ficoll,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(2-ethyl-2-oxazoline)-Tween, and-   Ficoll-Tween. In some specific embodiments, each phase of the    two-phase MPS is aqueous phase.

In yet another aspect, multi-phase system comprising two or more phasesis described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), poly(2-ethyl-2-oxazoline), poly(ethylene glycol), polyacrylamide,and Tween,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of poly(ethylene glycol)-Tween. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), polyacrylamide, andTween, respectively. In some specific embodiments, each phase of the MPSis aqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide-Tween,-   poly(methacrylic acid)-poly(ethylene glycol)-polyacrylamide-Tween,    and-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide-Tween. In some    specific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol),-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Tween,-   poly(methacrylic acid)-poly(ethylene glycol)-polyacrylamide,-   poly(methacrylic acid)-poly(ethylene glycol)-Tween,-   poly(methacrylic acid)-polyacrylamide-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Tween,-   poly(2-ethyl-2-oxazoline)-polyacrylamide-Tween, and-   poly(ethylene glycol)-polyacrylamide-Tween. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline),-   poly(methacrylic acid)-poly(ethylene glycol),-   poly(methacrylic acid)-Tween,-   poly(methacrylic acid)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-Tween,-   poly(ethylene glycol)-polyacrylamide, and-   polyacrylamide-Tween. In some specific embodiments, each phase of    the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(vinyl alcohol), poly(2-ethyl-2-oxazoline), poly(ethylene        glycol), polyacrylamide, and Tween,    -   with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofpoly(vinyl alcohol)-poly(ethylene glycol) and poly(ethyleneglycol)-Tween. In some embodiments, the MPS comprises a mixture ofaqueous and organic phases. In some embodiments, the MPS comprises allaqueous phases. In some embodiments, the MPS comprises all organicphases.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), polyacrylamide, andTween, respectively. In some specific embodiments, each phase of the MPSis aqueous phase.

In some embodiments, wherein the multi-phase system is a four-phasesystem and the phase components in the four phases are a combination ofphase components selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyacrylamide-Tween,-   poly(vinyl alcohol)-poly(ethylene glycol)-polyacrylamide-Tween, and-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-polyacrylamide-Tween.    In some specific embodiments, each phase of the MPS is aqueous    phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Tween,-   poly(vinyl alcohol)-poly(ethylene glycol)-polyacrylamide,-   poly(vinyl alcohol)-poly(ethylene glycol)-Tween,-   poly(vinyl alcohol)-polyacrylamide-Tween,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Tween,-   poly(2-ethyl-2-oxazoline)-polyacrylamide-Tween, and-   poly(ethylene glycol)-polyacrylamide-Tween. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline),-   poly(vinyl alcohol)-Tween,-   poly(vinyl alcohol)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-Tween,-   poly(ethylene glycol)-polyacrylamide, and-   polyacrylamide-Tween. In some specific embodiments, each phase of    the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(methacrylic acid), poly(2-ethyl-2-oxazoline), poly(ethylene        glycol), polyethyleneimine, and 1-O-Octyl-β-D-glucopyranoside,        with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of 1-O-Octyl-β-D-glucopyranoside-poly(ethyleneglycol). In some embodiments, the MPS comprises a mixture of aqueous andorganic phases. In some embodiments, the MPS comprises all aqueousphases. In some embodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), polyethyleneimine, and1-O-Octyl-β-D-glucopyranoside, respectively. In some specificembodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-1-O-Octyl-β-D-glucopyranoside,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-polyethyleneimine-1-O-Octyl-3-D-glucopyranoside,-   poly(methacrylic acid)-poly(ethylene    glycol)-polyethyleneimine-1-O-Octyl-β-D-glucopyranoside, and-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine-1-O-Octyl-3-D-glucopyranoside.    In some specific embodiments, each phase of the MPS is aqueous    phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol),-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-1-O-Octyl-β-D-glucopyranoside,-   poly(methacrylic acid)-poly(ethylene glycol)-polyethyleneimine,-   poly(methacrylic acid)-poly(ethylene    glycol)-1-O-Octyl-β-D-glucopyranoside,-   poly(methacrylic    acid)-polyethyleneimine-1-O-Octyl-β-D-glucopyranoside,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-poly(ethylene    glycol)-1-O-Octyl-β-D-glucopyranoside,-   poly(2-ethyl-2-oxazoline)-polyethyleneimine-1-O-Octyl-β-D-glucopyranoside,    and-   poly(ethylene    glycol)-polyethyleneimine-1-O-Octyl-β-D-glucopyranoside. In some    specific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline),-   poly(methacrylic acid)-poly(ethylene glycol),-   poly(methacrylic acid)-1-O-Octyl-β-D-glucopyranoside,-   poly(methacrylic acid)-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(2-ethyl-2-oxazoline)-polyethyleneimine,-   poly(2-ethyl-2-oxazoline)-1-O-Octyl-β-D-glucopyranoside,-   poly(ethylene glycol)-polyethyleneimine, and-   polyacrylamide-1-O-Octyl-β-D-glucopyranoside. In some specific    embodiments, each phase of the two-phase MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(vinyl alcohol), poly(2-ethyl-2-oxazoline), dextran, Ficoll,        and Pluronic,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofpoly(vinyl alcohol)-dextran, and dextran-Ficoll. In some embodiments,the MPS comprises a mixture of aqueous and organic phases. In someembodiments, the MPS comprises all aqueous phases. In some embodiments,the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), dextran, Ficoll, and Pluronic, respectively.In some specific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-dextran-Ficoll,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-dextran-Pluronic,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Ficoll-Pluronic,-   poly(vinyl alcohol)-dextran-Ficoll-Pluronic, and-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll-Pluronic. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-dextran,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Pluronic,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-dextran-Pluronic,-   poly(2-ethyl-2-oxazoline)-Ficoll-Pluronic,-   dextran-Ficoll-Pluronic,-   poly(vinyl alcohol)-dextran-Ficoll,-   poly(vinyl alcohol)-dextran-Pluronic, and-   poly(vinyl alcohol)-Ficoll-Pluronic. In some specific embodiments,    each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline),-   poly(vinyl alcohol)-Ficoll,-   poly(vinyl alcohol)-Pluronic,-   poly(2-ethyl-2-oxazoline)-dextran,-   poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(2-ethyl-2-oxazoline)-Pluronic,-   dextran-Pluronic, and-   Ficoll-Pluronic. In some specific embodiments, each phase of the MPS    is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting ofpoly(2-ethyl-2-oxazoline), poly(ethylene glycol), dextran, Ficoll, andCHAPS,

with the proviso that the multi-phase system does not include:

an aqueous three-phase system wherein the phase components in the threephases are a combination of poly(ethylene glycol)-dextran-Ficoll; or

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofdextran-Ficoll, poly(ethylene glycol)-Ficoll, and poly(ethyleneglycol)-dextran. In some embodiments, the MPS comprises a mixture ofaqueous and organic phases. In some embodiments, the MPS comprises allaqueous phases. In some embodiments, the MPS comprises all organicphases.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are poly(2-ethyl-2-oxazoline),poly(ethylene glycol), dextran, Ficoll, and CHAPS, respectively. In somespecific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran-CHAPS,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Ficoll-CHAPS,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll-CHAPS, and-   poly(ethylene glycol)-dextran-Ficoll-CHAPS. In some specific    embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-Ficoll,-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-CHAPS,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-Ficoll-CHAPS,-   poly(ethylene glycol)-dextran-CHAPS,-   poly(ethylene glycol)-Ficoll-CHAPS, and-   dextran-Ficoll-CHAPS. In some specific embodiments, each phase of    the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),-   poly(2-ethyl-2-oxazoline)-dextran,-   poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(2-ethyl-2-oxazoline)-CHAPS,-   poly(ethylene glycol)-CHAPS,-   dextran-CHAPS, and-   Ficoll-CHAPS. In some specific embodiments, each phase of the    two-phase MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting ofpoly(2-ethyl-2-oxazoline), Pluronic, dextran, Ficoll, and CHAPS,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of dextran-Ficoll. In some embodiments, the MPScomprises a mixture of aqueous and organic phases. In some embodiments,the MPS comprises all aqueous phases. In some embodiments, the MPScomprises all organic phases.

In some embodiments, the multi-phase system is a five-phase system andthe phase components in the five phases are poly(2-ethyl-2-oxazoline),Pluronic F68, dextran, Ficoll, and CHAPS, respectively. In some specificembodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are a combination of phasecomponents selected from the group consisting of

-   poly(2-ethyl-2-oxazoline)-Pluronic-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-Pluronic-dextran-CHAPS,-   poly(2-ethyl-2-oxazoline)-Pluronic-Ficoll-CHAPS,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll-CHAPS, and-   Pluronic-dextran-Ficoll-CHAPS. In some specific embodiments, each    phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(2-ethyl-2-oxazoline)-Pluronic-dextran,-   poly(2-ethyl-2-oxazoline)-Pluronic-Ficoll,-   poly(2-ethyl-2-oxazoline)-Pluronic-CHAPS,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll,-   poly(2-ethyl-2-oxazoline)-Ficoll-CHAPS,-   Pluronic-dextran-Ficoll-   Pluronic-dextran-CHAPS,-   Pluronic-Ficoll-CHAPS, and-   dextran-Ficoll-CHAPS. In some specific embodiments, each phase of    the MPS is aqueous phase.

In some embodiments, the multi-phase system is a two-phase system andthe phase components in the two phases are a combination of phasecomponents selected from the group consisting of

-   poly(2-ethyl-2-oxazoline)-Pluronic,-   poly(2-ethyl-2-oxazoline)-dextran,-   poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(2-ethyl-2-oxazoline)-CHAPS,-   Pluronic-dextran,-   Pluronic-Ficoll,-   Pluronic-CHAPS,-   dextran-CHAPS, and-   Ficoll-CHAPS. In some specific embodiments, each phase of the    two-phase MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), poly(2-ethyl-2-oxazoline), polyacrylamide, and Triton. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), polyacrylamide, and Triton, respectively. Insome specific embodiments, each phase of the MPS is aqueous phase.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(methacrylic acid)-poly(2-ethyl-2-oxazoline)-Triton,-   poly(methacrylic acid)-polyacrylamide-Triton, and-   poly(2-ethyl-2-oxazoline)-polyacrylamide-Triton. In some    embodiments, the multi-phase system is a two-phase system and the    phase components in the two phases are a combination of phase    components selected from the group consisting of    poly(methacrylic acid)-poly(2-ethyl-2-oxazoline),    poly(methacrylic acid)-polyacrylamide,    poly(methacrylic acid)-Triton,    poly(2-ethyl-2-oxazoline)-polyacrylamide,    poly(2-ethyl-2-oxazoline)-Triton, and    polyacrylamide-Triton. In some specific embodiments, each phase of    the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(vinylalcohol), poly(2-ethyl-2-oxazoline), dextran, and Ficoll,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofdextran-Ficoll, and dextran-poly(vinyl alcohol). In some embodiments,the MPS comprises a mixture of aqueous and organic phases. In someembodiments, the MPS comprises all aqueous phases. In some embodiments,the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), dextran, and Ficoll, respectively. In someembodiments, the multi-phase system is a three-phase system and thephase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-dextran,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Ficoll,-   poly(2-ethyl-2-oxazoline)-dextran-Ficoll, and-   poly(vinyl alcohol)-dextran-Ficoll. In some embodiments, the    multi-phase system is a two-phase system and the phase components in    the two phases are a combination of phase components selected from    the group consisting of    poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline),    poly(vinyl alcohol)-dextran-Ficoll,    poly(2-ethyl-2-oxazoline)-dextran, and    poly(2-ethyl-2-oxazoline)-Ficoll. In some specific embodiments, each    phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(vinylalcohol), poly(2-ethyl-2-oxazoline), polyacrylamide, and Pluronic,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of polyacrylamide-poly(vinyl alcohol). In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), polyacrylamide, and Pluronic, respectively.In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of

-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline)-Pluronic,-   poly(vinyl alcohol)-polyacrylamide-Pluronic, and-   poly(2-ethyl-2-oxazoline)-polyacrylamide-Pluronic. In some    embodiments, the multi-phase system is a two-phase system and the    phase components in the two phases are a combination of phase    components selected from the group consisting of-   poly(vinyl alcohol)-poly(2-ethyl-2-oxazoline),-   poly(vinyl alcohol)-Pluronic,-   poly(2-ethyl-2-oxazoline)-polyacrylamide,-   poly(2-ethyl-2-oxazoline)-Pluronic, and-   polyacrylamide-Pluronic. In some specific embodiments, each phase of    the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of dextran, Ficoll,hydroxyethyl cellulose, and Tween, with the proviso that the multi-phasesystem does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofdextran-Ficoll and dextran-Tween. In some embodiments, the MPS comprisesa mixture of aqueous and organic phases. In some embodiments, the MPScomprises all aqueous phases. In some embodiments, the MPS comprises allorganic phases.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are dextran, Ficoll,hydroxyethyl cellulose, and Tween, respectively. In some embodiments,the multi-phase system is a three-phase system and the phase componentsin the three phases are a combination of phase components selected fromthe group consisting of

-   dextran-Ficoll-hydroxyethyl cellulose,-   dextran-Ficoll-Tween,-   dextran-hydroxyethyl cellulose-Tween, and-   Ficoll-hydroxyethyl cellulose-Tween. In some embodiments, the    multi-phase system is a two-phase system and the phase components in    the two phases are a combination of phase components selected from    the group consisting of-   dextran-hydroxyethyl cellulose,-   Ficoll-hydroxyethyl cellulose,-   Ficoll-Tween, and-   hydroxyethyl cellulose-Tween. In some specific embodiments, each    phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of dextran, Ficoll,hydroxyethyl cellulose, and Triton, with the proviso that themulti-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofdextran-Ficoll and dextran-Triton. In some embodiments, the MPScomprises a mixture of aqueous and organic phases. In some embodiments,the MPS comprises all aqueous phases. In some embodiments, the MPScomprises all organic phases.

In some embodiments, the multi-phase system is a four-phase system andthe phase components in the four phases are dextran, Ficoll,hydroxyethyl cellulose, and Triton, respectively. In some embodiments,the multi-phase system is a three-phase system and the phase componentsin the three phases are a combination of phase components selected fromthe group consisting of

-   dextran-Ficoll-hydroxyethyl cellulose,-   dextran-Ficoll-Triton,-   dextran-hydroxyethyl cellulose-Triton, and-   Ficoll-hydroxyethyl cellulose-Triton. In some embodiments, the    multi-phase system is a two-phase system and the phase components in    the two phases are a combination of phase components selected from    the group consisting of-   dextran-hydroxyethyl cellulose,-   Ficoll-hydroxyethyl cellulose,-   Ficoll-Triton, and-   hydroxyethyl cellulose-Triton. In some specific embodiments, each    phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(methacrylic acid), poly(2-ethyl-2-oxazoline),poly(ethylene glycol), and poly(diallyldimethyl ammonium chloride). Insome embodiments, the multi-phase system is a four-phase system and thephase components in the four phases are poly(methacrylic acid),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), andpoly(diallyldimethyl ammonium chloride), respectively. In someembodiments, wherein the multi-phase system is a three-phase system andthe phase components in the three phases are a combination of phasecomponents selected from the group consisting of poly(methacrylicacid)-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),

-   poly(methacrylic    acid)-poly(2-ethyl-2-oxazoline)-poly(diallyldimethyl ammonium    chloride),-   poly(methacrylic acid)-poly(ethylene glycol)-poly(diallyldimethyl    ammonium chloride), and-   poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-poly(diallyldimethyl    ammonium chloride). In some embodiments, the multi-phase system is a    two-phase system and the phase components in the two phases are a    combination of phase components selected from the group consisting    of poly(methacrylic acid)-poly(2-ethyl-2-oxazoline, poly(methacrylic    acid)-poly(ethylene glycol), poly(methacrylic    acid)-poly(diallyldimethyl ammonium chloride),    poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),    poly(2-ethyl-2-oxazoline)-poly(diallyldimethyl ammonium chloride),    and poly(ethylene glycol)-poly(diallyldimethyl ammonium chloride).    In some specific embodiments, each phase of the MPS is aqueous    phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(methacrylic acid), poly(2-ethyl-2-oxazoline), Ficoll,and Triton. In some embodiments, the multi-phase system is a four-phasesystem and the phase components in the four phases are poly(methacrylicacid), poly(2-ethyl-2-oxazoline), Ficoll, and Triton, respectively. Insome embodiments, the multi-phase system is a three-phase system and thephase components in the three phases are a combination of phasecomponents selected from the group consisting of poly(methacrylicacid)-poly(2-ethyl-2-oxazoline)-Ficoll, poly(methacrylicacid)-poly(2-ethyl-2-oxazoline)-Triton, poly(methacrylicacid)-Ficoll-Triton, and poly(2-ethyl-2-oxazoline)-Ficoll-Triton. Insome embodiments, the multi-phase system is a two-phase system and thephase components in the two phases are a combination of phase componentsselected from the group consisting of poly(methacrylicacid)-poly(2-ethyl-2-oxazoline), poly(methacrylic acid)-Ficoll,poly(methacrylic acid)-Triton, poly(2-ethyl-2-oxazoline)-Ficoll,poly(2-ethyl-2-oxazoline)-Triton, and Ficoll-Triton. In some specificembodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(vinyl alcohol), poly(2-ethyl-2-oxazoline),poly(ethylene glycol), and poly(diallyldimethyl ammonium chloride), withthe proviso that the multi-phase system does not include: an aqueoustwo-phase system wherein the phase components in the two phases are acombination of poly(ethylene glycol)-poly(vinyl alcohol). In someembodiments, the multi-phase system is a four-phase system and the phasecomponents in the four phases are poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), poly(ethylene glycol), andpoly(diallyldimethyl ammonium chloride), respectively. In someembodiments, the multi-phase system is a three-phase system and thephase components in the three phases are a combination of phasecomponents selected from the group consisting of poly(vinylalcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol), poly(vinylalcohol)-poly(2-ethyl-2-oxazoline)-poly(diallyldimethyl ammoniumchloride), poly(vinyl alcohol)-poly(ethyleneglycol)-poly(diallyldimethyl ammonium chloride), andpoly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-poly(diallyldimethylammonium chloride). In some embodiments, the multi-phase system is atwo-phase system and the phase components in the two phases are acombination of phase components selected from the group consisting ofpoly(vinyl alcohol)-poly(2-ethyl-2-oxazoline, poly(vinylalcohol)-poly(diallyldimethyl ammonium chloride),poly(2-ethyl-2-oxazoline)-poly(ethylene glycol),poly(2-ethyl-2-oxazoline)-poly(diallyldimethyl ammonium chloride), andpoly(ethylene glycol)-poly(diallyldimethyl ammonium chloride). In somespecific embodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(2-ethyl-2-oxazoline), dextran, Ficoll, and Triton,with the proviso that the multi-phase system does not include: anaqueous two-phase system wherein the phase components in the two phasesare a combination selected from the group consisting of dextran-Ficolland dextran-Triton. In some embodiments, the multi-phase system is afour-phase system and the phase components in the four phases arepoly(2-ethyl-2-oxazoline), dextran, Ficoll, and Triton, respectively. Insome embodiments, the multi-phase system is a three-phase system and thephase components in the three phases are a combination of phasecomponents selected from the group consisting ofpoly(2-ethyl-2-oxazoline)-dextran-Ficoll,poly(2-ethyl-2-oxazoline)-dextran-Triton,poly(2-ethyl-2-oxazoline)-Ficoll-Triton, and dextran-Ficoll-Triton. Insome embodiments, the multi-phase system is a two-phase system and thephase components in the two phases are a combination of phase componentsselected from the group consisting of poly(2-ethyl-2-oxazoline)-dextran,poly(2-ethyl-2-oxazoline)-Ficoll, poly(2-ethyl-2-oxazoline)-Triton, andFicoll-Triton. In some specific embodiments, each phase of the MPS isaqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(2-ethyl-2-oxazoline), dextran, Ficoll, and CHAPS,with the proviso that the multi-phase system does not include: anaqueous two-phase system wherein the phase components in the two phasesare a combination of dextran-Ficoll. In some embodiments, themulti-phase system is a four-phase system and the phase components inthe four phases are poly(2-ethyl-2-oxazoline), dextran, Ficoll, andCHAPS, respectively. In some embodiments, the multi-phase system is athree-phase system and the phase components in the three phases are acombination of phase components selected from the group consisting ofpoly(2-ethyl-2-oxazoline)-dextran-Ficoll,poly(2-ethyl-2-oxazoline)-dextran-CHAPS,poly(2-ethyl-2-oxazoline)-Ficoll-CHAPS, and dextran-Ficoll-CHAPS. Insome embodiments, the multi-phase system is a two-phase system and thephase components in the two phases are a combination of phase componentsselected from the group consisting of poly(2-ethyl-2-oxazoline)-dextran,poly(2-ethyl-2-oxazoline)-Ficoll, dextran-CHAPS,poly(2-ethyl-2-oxazoline)-CHAPS, and Ficoll-CHAPS. In some specificembodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(acrylic acid), poly(ethylene glycol), andpoly(diallyldimethyl ammonium chloride), with the proviso that themulti-phase system does not include: an aqueous two-phase system whereinthe phase components in the two phases are a combination ofpoly(ethylene glycol)-poly(acrylic acid). In some embodiments, themulti-phase system is a three-phase system and the phase components inthe three phases are poly(acrylic acid), poly(ethylene glycol), andpoly(diallyldimethyl ammonium chloride), respectively. In someembodiments, the multi-phase system is a two-phase system and the phasecomponents in the two phases are a combination of phase componentsselected from the group consisting of poly(acrylicacid)-poly(diallyldimethyl ammonium chloride), and poly(ethyleneglycol)-poly(diallyldimethyl ammonium chloride). In some specificembodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(acrylic acid), polyacrylamide, and Triton. In someembodiments, the multi-phase system is a three-phase system and thephase components in the three phases are poly(acrylic acid),polyacrylamide, and Triton, respectively. In some embodiments, themulti-phase system is a two-phase system and the phase components in thetwo phases are a combination of phase components selected from the groupconsisting of poly(acrylic acid)-polyacrylamide, poly(acrylicacid)-Triton, and polyacrylamide-Triton. In some specific embodiments,each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(2-ethyl-2-oxazoline), dextran sulfate, andpoly(styrene sulfonic acid). In some embodiments, the multi-phase systemis a three-phase system and the phase components in the three phases arepoly(2-ethyl-2-oxazoline), dextran sulfate, and poly(styrene sulfonicacid), respectively. In some embodiments, the multi-phase system is atwo-phase system and the phase components in the two phases are acombination of phase components selected from the group consisting ofpoly(2-ethyl-2-oxazoline)-dextran sulfate,poly(2-ethyl-2-oxazoline)-poly(styrene sulfonic acid), and dextransulfate-poly(styrene sulfonic acid). In some specific embodiments, eachphase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(2-ethyl-2-oxazoline), dextran sulfate, andpoly(ethylene glycol), with the proviso that the multi-phase system doesnot include: an aqueous two-phase system wherein the phase components inthe two phases are a combination of poly(ethylene glycol)-dextransulfate. In some embodiments, the multi-phase system is a three-phasesystem and the phase components in the three phases arepoly(2-ethyl-2-oxazoline), dextran sulfate, and poly(ethylene glycol),respectively. In some embodiments, the multi-phase system is a two-phasesystem and the phase components in the two phases are a combination ofphase components selected from the group consisting ofpoly(2-ethyl-2-oxazoline)-dextran sulfate, andpoly(2-ethyl-2-oxazoline)-poly(ethylene glycol). In some specificembodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein each phase comprises a phase component,wherein the phase component is selected from the group consisting of apolymer, a surfactant, and a combination thereof; each of the two ormore phases has a different density and the phases, taken together,represent a density gradient; and the phases are phase-separated fromeach other, wherein the phase components are selected from the groupsconsisting of poly(vinyl alcohol), polyethyleneimine, andpoly(2-ethyl-2-oxazoline). In some embodiments, the multi-phase systemis a three-phase system and the phase components in the three phases arepoly(vinyl alcohol), polyethyleneimine, and poly(2-ethyl-2-oxazoline),respectively. In some embodiments, the multi-phase system is a two-phasesystem and the phase components in the two phases are a combination ofphase components selected from the group consisting of poly(vinylalcohol)-polyethyleneimine, poly(vinylalcohol)-poly(2-ethyl-2-oxazoline, andpoly(2-ethyl-2-oxazoline)-polyethyleneimine. In some specificembodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(vinylalcohol), polyethyleneimine, and poly(2-ethyl-2-oxazoline). In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(vinyl alcohol),polyethyleneimine, and poly(2-ethyl-2-oxazoline), respectively. In someembodiments, the multi-phase system is a two-phase system and the phasecomponents in the two phases are a combination of phase componentsselected from the group consisting of poly(vinylalcohol)-polyethyleneimine, poly(vinylalcohol)-poly(2-ethyl-2-oxazoline, andpoly(2-ethyl-2-oxazoline)-polyethyleneimine. In some specificembodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), poly(ethylene glycol), polyvinylpyrrolidone,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of poly(ethylene glycol)-polyvinylpyrrolidone.In some embodiments, the MPS comprises a mixture of aqueous and organicphases. In some embodiments, the MPS comprises all aqueous phases. Insome embodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(methacrylic acid),poly(ethylene glycol), and polyvinylpyrrolidone), respectively. In someembodiments, the multi-phase system is a two-phase system and the phasecomponents in the two phases are a combination of phase componentsselected from the group consisting of poly(methacrylicacid)-poly(ethylene glycol) and poly(methacrylicacid)-polyvinylpyrrolidone. In some specific embodiments, each phase ofthe MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), Ficoll, and Triton. In some embodiments, the MPS comprises amixture of aqueous and organic phases. In some embodiments, the MPScomprises all aqueous phases. In some embodiments, the MPS comprises allorganic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(methacrylic acid),Ficoll, and Triton, respectively. In some embodiments, the multi-phasesystem is a two-phase system and the phase components in the two phasesare a combination of phase components selected from the group consistingof poly(methacrylic acid)-Triton, poly(methacrylic acid)-Ficoll, andFicoll-Triton. In some specific embodiments, each phase of the MPS isaqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), polyacrylamide, and Zonyl. In some embodiments, the MPS comprisesa mixture of aqueous and organic phases. In some embodiments, the MPScomprises all aqueous phases. In some embodiments, the MPS comprises allorganic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(methacrylic acid),polyacrylamide, and Zonyl, respectively. In some embodiments, themulti-phase system is a two-phase system and the phase components in thetwo phases are a combination of phase components selected from the groupconsisting of poly(methacrylic acid)-polyacrylamide, poly(methacrylicacid)-Zonyl, and polyacrylamide-Zonyl. In some specific embodiments,each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof; each of the two or more phases has a differentdensity and the phases, taken together, represent a density gradient;and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), polyacrylamide, and N,N-dimethyldodecylamine N-oxide. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(methacrylic acid),polyacrylamide, and N,N-dimethyldodecylamine N-oxide, respectively. Insome embodiments, the multi-phase system is a two-phase system and thephase components in the two phases are a combination of phase componentsselected from the group consisting of poly(methacrylicacid)-polyacrylamide, poly(methacrylic acid)-N,N-dimethyldodecylamineN-oxide, and polyacrylamide-N,N-dimethyldodecylamine N-oxidel. In somespecific embodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(methacrylicacid), polyethyleneimine, and carboxy-polyacrylamide. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(methacrylic acid),polyethyleneimine, and carboxy-polyacrylamide, respectively. In someembodiments, the multi-phase system is a two-phase system and the phasecomponents in the two phases are a combination of phase componentsselected from the group consisting of poly(methacrylicacid)-polyethyleneimine, poly(methacrylic acid)-carboxy-polyacrylamide,and polyethyleneimine-carboxy-polyacrylamide. In some specificembodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(acrylic acid), poly(ethylene glycol), polyacrylamide,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of poly(acrylic acid)-poly(ethylene glycol).

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(acrylic acid),poly(ethylene glycol), and polyacrylamide, respectively. In someembodiments, the multi-phase system is a two-phase system and the phasecomponents in the two phases are a combination of phase componentsselected from the group consisting of poly(acrylic acid)-polyacrylamideand poly(ethylene glycol)-polyacrylamide. In some specific embodiments,each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(vinylalcohol), poly(2-ethyl-2-oxazoline), and dextran sulfate. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), and dextran sulfate, respectively. In someembodiments, the multi-phase system is a two-phase system and the phasecomponents in the two phases are a combination of phase componentsselected from the group consisting of poly(vinylalcohol)-poly(2-ethyl-2-oxazoline), poly(vinyl alcohol)-dextran sulfate,and poly(2-ethyl-2-oxazoline)-dextran sulfate. In some specificembodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting of poly(vinylalcohol), poly(2-ethyl-2-oxazoline), and chondroitin sulfate A. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(vinyl alcohol),poly(2-ethyl-2-oxazoline), and chondroitin sulfate A, respectively. Insome embodiments, the multi-phase system is a two-phase system and thephase components in the two phases are a combination of phase componentsselected from the group consisting of poly(vinylalcohol)-poly(2-ethyl-2-oxazoline), poly(vinyl alcohol)-chondroitinsulfate A, and poly(2-ethyl-2-oxazoline)-chondroitin sulfate A. In somespecific embodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(vinyl alcohol), poly(ethylene glycol), and dextran sulfate,    -   with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofpoly(vinyl alcohol)-poly(ethylene glycol) and poly(ethyleneglycol)-dextran sulfate. In some embodiments, the MPS comprises amixture of aqueous and organic phases. In some embodiments, the MPScomprises all aqueous phases. In some embodiments, the MPS comprises allorganic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(vinyl alcohol),poly(ethylene glycol), and dextran sulfate, respectively. In someembodiments, the multi-phase system is a two-phase system and the phasecomponents in the two phases are a combination of poly(vinylalcohol)-dextran sulfate. In some specific embodiments, each phase ofthe MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        poly(2-ethyl-2-oxazoline), dextran, and Triton, with the proviso        that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of dextran-Triton. In some embodiments, the MPScomprises a mixture of aqueous and organic phases. In some embodiments,the MPS comprises all aqueous phases. In some embodiments, the MPScomprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(2-ethyl-2-oxazoline),dextran, and Triton, respectively. In some embodiments, the multi-phasesystem is a two-phase system and the phase components in the two phasesare a combination of phase components selected from the group consistingof poly(2-ethyl-2-oxazoline)-dextran andpoly(2-ethyl-2-oxazoline)-Triton. In some specific embodiments, eachphase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting ofpoly(2-ethyl-2-oxazoline), Ficoll, and Brij. In some embodiments, theMPS comprises a mixture of aqueous and organic phases. In someembodiments, the MPS comprises all aqueous phases. In some embodiments,the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(2-ethyl-2-oxazoline),Ficoll, and Brij, respectively. In some embodiments, the multi-phasesystem is a two-phase system and the phase components in the two phasesare a combination of phase components selected from the group consistingof poly(2-ethyl-2-oxazoline)-Ficoll, poly(2-ethyl-2-oxazoline)-Brij, andFicoll-Brij. In some specific embodiments, each phase of the MPS isaqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

each phase comprises a phase component, wherein the phase component isselected from the group consisting of a polymer, a surfactant, and acombination thereof;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient; and

the phases are phase-separated from each other, wherein the phasecomponents are selected from the groups consisting ofpoly(2-ethyl-2-oxazoline), Ficoll, and Triton. In some embodiments, theMPS comprises a mixture of aqueous and organic phases. In someembodiments, the MPS comprises all aqueous phases. In some embodiments,the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are poly(2-ethyl-2-oxazoline),Ficoll, and Triton, respectively. In some embodiments, the multi-phasesystem is a two-phase system and the phase components in the two phasesare a combination of phase components selected from the group consistingof poly(2-ethyl-2-oxazoline)-Ficoll, poly(2-ethyl-2-oxazoline)-Triton,and Ficoll-Triton. In some specific embodiments, each phase of the MPSis aqueous phase.

In yet another aspect, a multi-phase system comprising three phases isdescribed, wherein each phase comprises a phase component, wherein thephase component is selected from the group consisting of a polymer, asurfactant, and a combination thereof;

each of the phases has a different density and the phases, takentogether, represent a density gradient;

the phases are phase-separated from each other; and

the phase components in the three phases are poly(ethylene glycol) or aco- or ter-polymer thereof, polyvinylpyrrolidone or a co- or ter-polymerthereof, and dextran, respectively. In some embodiments, the MPScomprises a mixture of aqueous and organic phases. In some embodiments,the MPS comprises all aqueous phases. In some embodiments, the MPScomprises all organic phases.

In some embodiments, a multi-phase system comprising two or more phasesis described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        dextran, Ficoll, and Triton,    -   with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination selected from the group consisting ofdextran-Ficoll and dextran-Triton.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are dextran, Ficoll, andTriton, respectively. In some embodiments, the multi-phase system is atwo-phase system and the phase components in the two phases are acombination of Ficoll-Triton. In some specific embodiments, each phaseof the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        dextran, polyvinylpyrrolidone, and        poly(2-acrylamido-2-methyl-1-propanesulfonic acid),

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of dextran-polyvinylpyrrolidone. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are dextran,polyvinylpyrrolidone, and poly(2-acrylamido-2-methyl-1-propanesulfonicacid), respectively. In some embodiments, the multi-phase system is atwo-phase system and the phase components in the two phases are acombination of phase components selected from the group consisting ofpoly(2-acrylamido-2-methyl-1-propanesulfonic acid-dextran andpoly(2-acrylamido-2-methyl-1-propanesulfonic acid-polyvinylpyrrolidone.In some specific embodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        dextran, hydroxyethyl cellulose, and Triton,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of phase components selected from the groupconsisting of dextran-Triton and hydroxyethyl cellulose-Triton. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are dextran, hydroxyethylcellulose, and Triton, respectively. In some embodiments, themulti-phase system is a two-phase system and the phase components in thetwo phases are a combination of hydroxyethyl cellulose-dextran. In somespecific embodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a multi-phase system comprising two or morephases is described, wherein

-   -   each phase comprises a phase component, wherein the phase        component is selected from the group consisting of a polymer, a        surfactant, and a combination thereof;    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other, wherein the        phase components are selected from the groups consisting of        Ficoll, hydroxyethyl cellulose, and Triton,

with the proviso that the multi-phase system does not include:

an aqueous two-phase system wherein the phase components in the twophases are a combination of hydroxyethyl cellulose-Triton. In someembodiments, the MPS comprises a mixture of aqueous and organic phases.In some embodiments, the MPS comprises all aqueous phases. In someembodiments, the MPS comprises all organic phases.

In some embodiments, the multi-phase system is a three-phase system andthe phase components in the three phases are Ficoll, hydroxyethylcellulose, and Triton, respectively. In some embodiments, themulti-phase system is a two-phase system and the phase components in thetwo phases are a combination of phase components selected from the groupconsisting of hydroxyethyl cellulose-Ficoll and Ficoll-Triton. In somespecific embodiments, each phase of the MPS is aqueous phase.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein

-   -   the first phase comprises poly(diallyldimethyl ammonium        chloride);    -   the second phase comprises a phase component selected from the        group consisting of poly(methacrylic acid), poly(acrylic acid),        poly(vinyl alcohol), poly(2-ethyl-2-oxazoline), and        poly(ethylene glycol);    -   each of the two or more phases has a different density and the        phases, taken together, represent a density gradient; and    -   the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein

the first phase comprises chondroitin sulfate A;

the second phase comprises a phase component selected from the groupconsisting of poly(methacrylic acid), poly(vinyl alcohol), andpoly(2-ethyl-2-oxazoline);

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein

the first phase comprises poly(propylene glycol);

the second phase comprises a phase component selected from the groupconsisting of poly(methacrylic acid) and polyacrylamide;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein

the first phase comprises poly(styrene sulfonic acid);

the second phase comprises a phase component selected from the groupconsisting of poly(2-ethyl-2-oxazoline) and dextran sulfate;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein

the first phase comprises polyallylamine;

the second phase comprises a phase component selected from the groupconsisting of Tween, Triton, dextran sulfate, and Brij;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein

each of the first and second phases comprises a phase component;

the phase components in the two phases are a combination of phasecomponents selected from the group consisting ofdiethylaminoethyl-dextran-poly(acrylic acid),carboxy-polyacrylamide-poly(vinyl alcohol), methyl cellulose-Ficoll,Zonyl-dextran, Triton X-100-poly(acrylic acid), and sodium dodecylsulfate-poly(acrylic acid),

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein

the first phase comprises alginic acid;

the second phase comprises a phase component selected from the groupconsisting of poly(acrylic acid), and poly(propylene glycol);

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein

the first phase comprises (hydroxypropyl)methyl cellulose;

the second phase comprises a phase component selected from the groupconsisting of poly(diallyldimethyl ammonium chloride), andpoly(propylene glycol);

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases, wherein

the first phase comprises nonylphenol polyoxyethylene;

the second phase comprises a phase component selected from the groupconsisting of poly(methacrylic acid), and dextran;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In yet another aspect, a two-phase system comprising a first and secondphases is described, wherein the first phase comprises sodium cholate;

the second phase comprises a phase component selected from the groupconsisting of poly(methacrylic acid), and dextran sulfate;

each of the two or more phases has a different density and the phases,taken together, represent a density gradient, and

the phases are phase-separated from each other.

In some embodiments, the MPS is organic. Each phase of the organic MPScomprises an organic solvent and a phase component as defined herein.The organic multi-phase system comprising at least a first and secondnon-aqueous phases, wherein

-   -   the first phase is non-aqueous and comprise a first organic        solvent and a first phase component;    -   the second phase is non-aqueous and comprises a second organic        solvent and a second phase component;    -   the first and second phases are in contact and phase-separated        from each other;    -   the phase component is selected from the group consisting of a        polymer, a surfactant, and a combination thereof; and

each of the first and second phases has a different density and thephases, taken together, represent a density gradient. Non-limitingexamples of the first and second solvents include liquid polymer,dichloromethane, THF, ethanol, supercritical CO₂, fuel, and lubricant.The first and the second solvents can be the same or different. In somespecific embodiments, the first and second solvents are eachdichloromethane. In some embodiments, the organic MPS comprises phasecomponents selected from the group consisting of poly(bisphenol Acarbonate), polydimethylsiloxane, polystyrene, poly(4-vinylpyridine),poly(2-ethyl-2-oxazoline), polycaprolactone. In some embodiments, themulti-phase system is a six-phase system and the phase components in thesix phases are poly(bisphenol A carbonate), polydimethylsiloxane,polystyrene, poly(4-vinylpyridine), poly(2-ethyl-2-oxazoline),polycaprolactone, respectively. In some embodiments, the multi-phasesystem is a five-phase system and the phase components in the fivephases are a combination of phase components selected from the groupconsisting of

-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-polycaprolactone,-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-poly(2-ethyl-2-oxazoline)-poly    caprolactone,-   poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,-   poly(bisphenol A    carbonate)-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,    and-   polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone.    In some embodiments, the multi-phase system is a four-phase system    and the phase components in the four phases are a combination of    phase components selected from the group consisting of-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-poly(4-vinylpyridine),-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-poly(2-ethyl-2-oxazoline),-   poly(bisphenol A    carbonate)-polydimethylsiloxane-polystyrene-polycaprolactone,-   poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),-   poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(4-vinylpyridine)polycaprolactone,-   poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(2-ethyl-2-oxazoline)-polycaprolactone,-   poly(bisphenol A    carbonate)-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),-   poly(bisphenol A    carbonate)-polystyrene-poly(4-vinylpyridine)-polycaprolactone,-   poly(bisphenol A    carbonate)-polystyrene-poly(2-ethyl-2-oxazoline)-polycaprolactone,-   poly(bisphenol A    carbonate)-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,-   polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),-   polydimethylsiloxane-polystyrene-poly(4-vinylpyridine)-polycaprolactone,-   polydimethylsiloxane-polystyrene-poly(2-ethyl-2-oxazoline)-polycaprolactone,-   polydimethylsiloxane-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone,    and-   polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone.    In some embodiments, the multi-phase system is a three-phase system    and the phase components in the three phases are a combination of    phase components selected from the group consisting of-   poly(bisphenol A carbonate)-polydimethylsiloxane-polystyrene,-   poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(4-vinylpyridine),-   poly(bisphenol A    carbonate)-polydimethylsiloxane-poly(2-ethyl-2-oxazoline,-   poly(bisphenol A carbonate)-polydimethylsiloxane-polycaprolactone,-   poly(bisphenol A carbonate)-polystyrene-poly(4-vinylpyridine),-   poly(bisphenol A carbonate)-polystyrene-poly(2-ethyl-2-oxazoline),-   poly(bisphenol A carbonate)-polystyrene-polycaprolactone,-   poly(bisphenol A    carbonate)-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),-   poly(bisphenol A carbonate)-poly(4-vinylpyridine)-polycaprolactone,-   poly(bisphenol A    carbonate)-poly(2-ethyl-2-oxazoline)-polycaprolactone,-   polydimethylsiloxane-polystyrene-poly(4-vinylpyridine),-   polydimethylsiloxane-polystyrene-poly(2-ethyl-2-oxazoline),-   polydimethylsiloxane-polystyrene-polycaprolactone,-   polydimethylsiloxane-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),-   polydimethylsiloxane-poly(4-vinylpyridine)-polycaprolactone,-   polydimethylsiloxane-poly(2-ethyl-2-oxazoline)-polycaprolactone,-   polystyrene-poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),-   polystyrene-poly(4-vinylpyridine)-polycaprolactone,-   polystyrene-poly(2-ethyl-2-oxazoline)-polycaprolactone, and-   poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline)-polycaprolactone. In    some embodiments, the multi-phase system is a two-phase system and    the phase components in the two phases are a combination of phase    components selected from the group consisting of-   poly(bisphenol A carbonate)-polydimethylsiloxane,-   poly(bisphenol A carbonate)-polystyrene,-   poly(bisphenol A carbonate)-poly(4-vinylpyridine),-   poly(bisphenol A carbonate)-poly(2-ethyl-2-oxazoline),-   poly(bisphenol A carbonate)-polycaprolactone,-   polydimethylsiloxane-polystyrene,-   polydimethylsiloxane-poly(4-vinylpyridine),-   polydimethylsiloxane-poly(2-ethyl-2-oxazoline),-   polydimethylsiloxane-polycaprolactone,-   polystyrene-poly(4-vinylpyridine),-   polystyrene-poly(2-ethyl-2-oxazoline),-   polystyrene-polycaprolactone,-   poly(4-vinylpyridine)-poly(2-ethyl-2-oxazoline),-   poly(4-vinylpyridine)-polycaprolactone, and-   poly(2-ethyl-2-oxazoline)-polycaprolactone.

In some embodiments, the organic multi-phase system is a two-phasesystem, wherein the first phase comprises poly(bisphenol A carbonate),the second phase comprises a phase component selected from the groupconsisting of polysulfone, polyvinylpyrrolidone, poly(methylmethacrylate-co-methacrylic acid), poly(methyl methacrylate), Tween,poly(tetrahydrofuran), poly(propylene glycol), poly(ethylene glycol),and poly(vinyl acetate). In some embodiments, the organic multi-phasesystem is a two-phase system, wherein the first phase comprisespolysulfone, the second phase comprises a phase component selected fromthe group consisting of polydimethylsiloxane, polystyrene,poly(4-vinylpyridine), polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline),polyethyleneimine, poly(methyl methacrylate-co-methacrylic acid),poly(methyl methacrylate), poly(tetrahydrofuran), poly(propyleneglycol), poly(ethylene glycol), poly(vinyl acetate), andpolycaprolactone. In some embodiments, the organic multi-phase system isa two-phase system, wherein the first phase comprisespolydimethylsiloxane, the second phase comprises a phase componentselected from the group consisting of polyvinylpyrrolidone,polyethyleneimine, poly(methyl methacrylate-co-methacrylic acid),poly(methyl methacrylate), Tween, poly(tetrahydrofuran), poly(ethyleneglycol), and poly(vinyl acetate).

In some embodiments, the organic multi-phase system is a two-phasesystem, wherein the first phase comprises polystyrene, the second phasecomprises a phase component selected from the group consisting ofpolyvinylpyrrolidone, polyethyleneimine, poly(methylmethacrylate-co-methacrylic acid), poly(methyl methacrylate),poly(tetrahydrofuran), Tween, poly(ethylene glycol), and poly(vinylacetate). In some embodiments, the organic multi-phase system is atwo-phase system, wherein the first phase comprisespoly(4-vinylpyridine), the second phase comprises a phase componentselected from the group consisting of polyethyleneimine, poly(methylmethacrylate-co-methacrylic acid), poly(methyl methacrylate),poly(tetrahydrofuran), Tween, poly(propylene glycol), poly(ethyleneglycol), and poly(vinyl acetate).

In some embodiments, the organic multi-phase system is a two-phasesystem, wherein the first phase comprises polyvinylpyrrolidone, thesecond phase comprises a phase component selected from the groupconsisting of poly(tetrahydrofuran), poly(propylene glycol),poly(ethylene glycol), and poly(vinyl acetate). In some embodiments, theorganic multi-phase system is a two-phase system, wherein the firstphase comprises poly(2-ethyl-2-oxazoline), the second phase comprises aphase component selected from the group consisting ofpoly(tetrahydrofuran), poly(propylene glycol), poly(ethylene glycol),and poly(vinyl acetate). In some embodiments, the organic multi-phasesystem is a two-phase system, wherein the first phase comprisespolyethyleneimine, the second phase comprises a phase component selectedfrom the group consisting of poly(ethylene glycol), polycaprolactone,and poly(vinyl acetate). In some embodiments, the organic multi-phasesystem is a two-phase system, wherein the first phase comprisespolycaprolactone, the second phase comprises a phase component selectedfrom the group consisting of poly(methyl methacrylate-co-methacrylicacid) and poly(methyl methacrylate).

In some embodiments, the MPS comprises two or more liquid polymer phases(solvent free phases; see Experimental Section for the acronym of thepolymers). In some embodiments, the liquid polymer MPS is a two phaseliquid polymer MPS selected from the group consisting of: PPG-PEG;PPG-PL; PPG-PDMS; PPG-PBD; PEG-PL; PEG-PDMS; PEG-PEVE; PEG-PBD; PL-PDMS;PL-PBD; PDMS-PEVE; PDMS-PBD; and PEVE-PBD.

In some embodiments, the liquid polymer MPS is a three phase liquidpolymer MPS selected from the group consisting of: PPG-PEG-PL;PPG-PDMS-PBD; PPG-PEG-PBD; PEG-PEVE-PBD; PEG-PEVE-PDMS; PPG-PEG-PDMS;PEG-PL-PDMS; PEG-PL-PBD; PL-PDMS-PBD; and PDMS-PEVE-PBD.

In some embodiments, the liquid polymer MPS is a four phase liquidpolymer MPS selected from the group consisting of: PPG-PEG-PL-PDMS;PPG-PEG-PL-PBD; PPG-PEG-PDMS-PBD; PPG-PL-PDMS-PBD; and PEG-PL-PDMS-PBD.

In some embodiments, the liquid polymer MPS is a five phase liquidpolymer MPS having the composition of PPG-PEG-PL-PDMS-PBD.

In some embodiments, the MPS has is a two-phase system wherein thecombination of reagents of the phases is selected from the groupconsisting of PVPNO-PA, PVPNO-PMAA, PVPNO-PAA, PVPNO-PVA, PVPNO-PEOZ,PVPNO-PEG, PVPNO-HEC, PVPNO-Dextran, PVPNO-Ficoll, PVPNO-PEI,PVPNO-Tween, and PVPNO-PVP (see Experimental Section for the acronym ofthe polymers).

In some embodiments, the MPS has is a three-phase system wherein thecombination of reagents of the phases is selected from the groupconsisting of PEOZ-PEG-PVPNO, PEOZ-PEI-PVPNO, PEOZ-PA-PVPNO,PEOZ-PMAA-PVPNO, PEG-PEI-PVPNO, PEG-PMAA-PVPNO, PEG-PA-PVPNO,PEI-PA-PVPNO, PEI-PMAA-PVPNO, PA-PMAA-PVPNO, PEOZ-PEG-PVPNO,PEOZ-TWEEN-PVPNO, PEOZ-PA-PVPNO, PEOZ-PMAA-PVPNO, PEG-TWEEN-PVPNO,TWEEN-PA-PVPNO, TWEEN-PMAA-PVPNO, PA-PMAA-PVPNO, PEG-PA-PVPNO, andPEG-PMAA-PVPNO (see Experimental Section for the acronym of thepolymers).

In some embodiments, the MPS has is a four-phase system wherein thecombination of reagents of the phases is selected from the groupconsisting of PEOZ-PEG-PEI-PVPNO, PEOZ-PEG-PA-PVPNO, PEOZ-PEI-PA-PVPNO,PEOZ-PEI-PMAA-PVPNO, PEOZ-PA-PMAA-PVPNO, PEG-PEI-PA-PVPNO,PEG-PEI-PMAA-PVPNO, PEG-PA-PMAA-PVPNO, PEI-PA-PMAA-PVPNO,PEOZ-PEG-PMAA-PVPNO, PEOZ-PEG-PA-PVPNO, PEOZ-PEG-TWEEN-PVPNO,PEOZ-TWEEN-PA-PVPNO, PEOZ-TWEEN-PMAA-PVPNO, PEOZ-PA-PMAA-PVPNO,PEG-TWEEN-PA-PVPNO, PEG-TWEEN-PMAA-PVPNO, PEG-PA-PMAA-PVPNO, andTWEEN-PA-PMAA-PVPNO (see Experimental Section for the acronym of thepolymers).

In some embodiments, the MPS has is a five-phase system wherein thecombination of reagents of the phases is selected from the groupconsisting of PEG-PEI-PA-PMAA-PVPNO, PEOZ-PEI-PA-PMAA-PVPNO,PEOZ-PEG-PA-PMAA-PVPNO, PEOZ-PEG-PEI-PMAA-PVPNO, PEOZ-PEG-PEI-PA-PVPNO,PEOZ-PEG-PEI-PA-PMAA, PEG-TWEEN-PA-PMAA-PVPNO, PEOZ-TWEEN-PA-PMAA-PVPNO,PEOZ-PEG-TWEEN-PMAA-PVPNO, PEOZ-PEG-TWEEN-PA-PVPNO, andPEOZ-PEG-TWEEN-PA-PMAA (see Experimental Section for the acronym of thepolymers).

In some embodiments, the MPS has is a six-phase system wherein thecombination of reagents of the phases is selected from the groupconsisting of PEOZ-PEG-PEI-PA-PMAA-PVPNO andPEOZ-PEG-TWEEN-PA-PMAA-PVPNO (see Experimental Section for the acronymof the polymers).

In some embodiments, the MPS system has the composition selected formthe compositions disclosed in Tables S2-S6.

Experimental Section

Chemicals

Allura Red, alginic acid sodium salt, Brij 35, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), chondroitin sulfate A,dextran sulfate sodium salt, ethyl orange, Ficoll, (hydroxypropyl)methylcellulose, poly(2-acrylamido-2-methyl-1-propanesulfonic acid),poly(2-ethyl-2-oxazoline), polyacrylamide, poly(diallyldimethylammoniumchloride), poly(ethylene glycol), polyethyleneimine, poly(methacrylicacid sodium salt), poly(propylene glycol), polyvinylpyrrolidone, sodiumcholate, Tween 20, Pluronic F68 and Zonyl were purchased fromSigma-Aldrich. Poly(acrylic acid), poly(allylamine hydrochloride),poly(styrenesulfonic acid sodium salt), poly(2-vinylpyridine-N-oxide),and poly(vinyl alcohol) were obtained from Polysciences. Dextran waspurchased from Spectrum Chemical. Diethylaminoethyl-dextranhydrochloride was purchased from MP Biomedicals. Carboxy-modifiedpolyacrylamide, hydroxyethyl cellulose, methyl cellulose were purchasedfrom Scientific Polymer Products. DOWFAX 2A1 was purchased from DowChemicals. N, N-dimethyldodecylamine N-oxide was purchased from Fluka.N-octyl-β-D-glucopyranoside was purchased from Alfa Aesar. Sodiumdodecyl sulfate was purchased from J. T. Baker. Triton X-100 waspurchased from Calbiochem. All chemicals were used without furtherpurification.

Experimental Designs

Binary mixtures of twenty-three polymers and eleven surfactants wereinvestigated. Applicants then systematically examined potential three-,four-, five-, and six-solution mixtures based on the results of thebinary immiscibility screen to identify systems that exhibitedsegregation into multiple phases. For example, poly(propyleneglycol)-poly(methacrylic acid), poly(propylene glycol)-polyacrylamide,and poly(methacrylic acid)-polyacrylamide ATPS were combined to producea poly(propylene glycol)-polyacrylamide-poly(methacrylic acid)three-phase MPS.

Stock Solutions

Applicants prepared stock solutions of polymers in Milli-Q water at highconcentrations, between 1-40% (w/v) without adding salts or titratingthe pH. Applicants characterized the density, refractive index, andosmolality of each stock solution using oscillating U-tube densitometry(Anton Paar DM35N), refractometry (Bausch & Lomb Abbe Refractometer),and freezing point depression osmometry (Advanced Instruments Model 3000MicroOsmometer). The results of these characterizations are given inTable S1. The densities for stock solutions of polyacrylamide andpoly(2-acrylamido-2-methyl-1-propanesulfonic acid) were determined bymeasuring the densities of three lower concentrations and determiningthe linear fit to the resulting line.

TABLE S1 Properties of the polymer stock solutions: average molecularweight (Da), concentration (% wt/vol), density (g/cm³), osmolality(mOsm/kg), and refractive index; “n.d.” stands for “not determined”, andan asterisk (*) refers to densities that were calculated, rather thanmeasured. avg. % density osmolality refractive Polymer MW (Da) (wt/vol)(g/cm³) (mOsm/kg) index 1 poly(methacrylic acid) 5,000 40 1.279 n.d.1.4250 2 poly(acrylic acid) 450,000 10 1.035  28 1.3445 3 poly(vinylalcohol) 3,000 10 1.022  84 1.3635 4 poly(2-ethyl-2-oxazoline) 200,00035 1.059 n.d. 1.3865 5 poly(ethylene glycol) 20,000 40 1.069 n.d. 1.38606 dextran 500,000 30 1.101 286 1.3700 7 Ficoll 400,000 40 1.130 2241.3860 8 polyacrylamide 10,000 40  1.149 * n.d. 1.4130 9poly(diallyldimethylammonium 400,000 20 1.044 1241  1.3730 chloride) 10dextran sulfate 500,000 20 1.103 539 1.3540 11 Chondroitin sulfate A25,000 10 1.044 325 1.3460 12 polyethyleneimine 25,000 30 1.037 7451.3890 13 polyvinylpyrrolidone 55,000 20 1.038 782 1.3635 14poly(propylene glycol) 425 40 1.029 n.d. 1.3860 15poly(2-acrylamido-2-methyl-1- 2,000,000 15  1.042 * 353 1.3520propanesulfonic acid) 16 poly(styrenesulfonic acid) 75,000 30 1.100 n.d.1.3975 17 diethylaminoethyl-dextran 500,000 10 1.028 185 1.3460 18polyallylamine 60,000 20 1.052 826 1.3710 19 alginic acid 240,000 21.010  50 1.3345 20 (hydroxypropyl)methyl cellulose 10,000 2 1.003  71.3345 21 carboxy-polyacrylamide 200,000 6 1.018  93 1.3410 22hydroxyethyl cellulose 90,000 2 1.004  24 1.3340 23 methyl cellulose86,000 1 1.001  −6 1.3325

Example 1. Preparation of Multi-Phase Systems

Biphasic Separation

To perform the initial two-phase polymer immiscibility screens,Applicants added equal volumes (150 μL) of each polymer solution into amicrocentrifuge tube, vortexed the tube for 30 seconds to thoroughly mixthe solutions, and accelerated segregation into phases by centrifugationat 2,000×g for 10 minutes. In some instances, Applicants observed anacute volumetric rearrangement of a single layer of the aqueoustwo-phase system (ATPS). This is a well-understood phenomenon thatresults when the initial concentrations of the system are near the nodeof the tie-line of the phase diagram that characterizes the set ofmixtures for a polymer/polymer system. For such ATPS, Applicantsmodified either the volume ratio or diluted the polymer stock solutionin order to verify that phase separation occurred. From theinvestigation of 34 unique water-soluble polymers and surfactants,Applicants identified numerous unreported ATPS and confirmed those thathave been previously described by others (Table S2).

Results and Discussion

Applicants generated a series of miscibility profiles for each reagentby assigning a 34-component vector describing the results from alltwo-component mixtures that include the reagent. The vector has values‘0’ for mixtures that resulted in homogeneous solutions (miscible, shownin light gray square), ‘1’ for mixtures that resulted in a precipitateor a gel (incompatible, shown in black square), and ‘2’ for mixturesthat resulted in phase separation (immiscible, shown in dark graysquare).

Applicants can compare the miscibility profiles of N reagents, andclusters of reagents, by analyzing the magnitudes of each vector and thedistances between vectors in N-dimensional space: a small distancebetween vectors indicates similar miscibility profiles. Applicantsordered the reagents in our AMS matrix according to this vector analysis(FIG. 1).

Using this approach to ordering, Applicants identified several patternsbased on similarities in miscibility in two-component mixtures: neutral,branched polysaccharides (numbers 3 and 4), acrylic acids (4 and 5),cationic species (10, 12, and 13), hydrophobic species incorporatingethylene oxide units (14-18), and anionic species (21, 22, 24, 26-29)are clustered by patterns of miscibility using this analysis.

Applicants observed phase separation in formerly characterized two-phasesystems, but Applicants also identified numerous previously unreportedATPS. Examples of these new systems includepolyvinylpyrrolidone-poly(methacrylic acid), poly(styrenesulfonicacid)-alginic acid, and poly(vinyl alcohol)-carboxy-polyacrylamide. Thecomplete two-phase ATPS results are shown in Table S2, while the listsof three-, four-, five-, and six-phase MPS are shown in Table S4, TableS5, and Table S6, respectively.

Images of multiphase polymer systems are shown in FIGS. 2(A)-2(C). FIG.2(A) shows a three-phase system comprising poly(propyleneglycol)-polyacrylamide-poly(methacrylic acid). FIG. 2(B) shows afour-phase system comprising poly(vinylalcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-polyacrylamide.FIG. 2(C) shows a five-phase system comprising poly(vinylalcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran-Ficoll.Allura Red, a dye, was added to highlight the interfaces of each polymersystem; the color balance and contrast of the inset image were modifiedto further differentiate the second and third layers of the five-phasepolymer system, which are marked by arrows. The most complex AMPS fromthis survey were two systems composed of five layers: (i)polyethyleneimine-poly(2-ethyl-2-oxazoline)-poly(ethyleneglycol)-polyacrylamide-poly(methacrylic acid) and (ii) poly(vinylalcohol)-poly(2-ethyl-2-oxazoline)-poly(ethylene glycol)-dextran-Ficoll(FIG. 2C).

TABLE S2 List of new and previously reported aqueous MPS found by thisstudy. NEW (1) Count Polymers OLD (0) 1 poly(2-ethyl-2-oxazoline)poly(methacrylic acid) 1 2 poly(2-ethyl-2-oxazoline) poly(vinyl alcohol)1 3 poly(ethylene glycol) poly(methacrylic acid) 1 4 poly(ethyleneglycol) poly(acrylic acid) 0 5 poly(ethylene glycol) poly(vinyl alcohol)0 6 poly(ethylene glycol) poly(2-ethyl-2-oxazoline) 1 7 dextranpoly(vinyl alcohol) 0 8 dextran poly(2-ethyl-2-oxazoline) 1 9 dextranpoly(ethylene glycol) 0 10 Ficoll poly(methacrylic acid) 1 11 Ficollpoly(vinyl alcohol) 1 12 Ficoll poly(2-ethyl-2-oxazoline) 1 13 Ficollpoly(ethylene glycol) 0 14 Ficoll dextran 0 15 polyacrylamidepoly(methacrylic acid) 1 16 polyacrylamide poly(acrylic acid) 1 17polyacrylamide poly(vinyl alcohol) 0 18 polyacrylamidepoly(2-ethyl-2-oxazoline) 1 19 polyacrylamide poly(ethylene glycol) 1 20poly(diallyldimethyl ammonium chloride poly(methacrylic acid) 1 21poly(diallyldimethyl ammonium chloride poly(acrylic acid) 1 22poly(diallyldimethyl ammonium chloride poly(vinyl alcohol) 1 23poly(diallyldimethyl ammonium chloride poly(2-ethyl-2-oxazoline) 1 24poly(diallyldimethyl ammonium chloride poly(ethylene glycol) 1 25dextran sulfate poly(vinyl alcohol) 1 26 dextran sulfatepoly(2-ethyl-2-oxazoline) 1 27 dextran sulfate poly(ethylene glycol) 028 chondroitin sulfate A poly(methacrylic acid) 1 29 chondroitin sulfateA poly(vinyl alcohol) 1 30 chondroitin sulfate Apoly(2-ethyl-2-oxazoline) 1 31 polyethyleneimine poly(methacrylic acid)1 32 polyethyleneimine poly(2-ethyl-2-oxazoline) 1 33 polyethyleneiminepoly(ethylene glycol) 1 34 polyethyleneimine Ficoll 1 35polyethyleneimine polyacrylamide 1 36 polyvinylpyrrolidonepoly(methacrylic acid) 1 37 polyvinylpyrrolidone poly(ethylene glycol) 038 polyvinylpyrrolidone dextran 0 39 poly(propylene glycol)poly(methacrylic acid) 1 40 poly(propylene glycol) dextran 0 41poly(propylene glycol) polyacrylamide 1 42poly(2-acrylamido-2-methyl-1-propanesulfonic acid) dextran 1 43poly(2-acrylamido-2-methyl-1-propanesulfonic acid) polyvinylpyrrolidone1 44 poly(styrene sulfonic acid) poly(2-ethyl-2-oxazoline) 1 45poly(styrene sulfonic acid) dextran sulfate 1 46diethylaminoethyl-dextran poly(acrylic acid) 1 47 polyallylamine dextransulfate 1 48 alginic acid poly(acrylic acid) 1 49 alginic acidpoly(propylene glycol) 1 50 (hydroxypropyl)methyl cellulosepoly(diallyldimethyl ammonium chloride 1 51 (hydroxypropyl)methylcellulose poly(propylene glycol) 1 52 carboxy-polyacrylamidepoly(methacrylic acid) 1 53 carboxy-polyacrylamide poly(vinyl alcohol) 154 carboxy-polyacrylamide polyethyleneimine 1 55 hydroxyethyl cellulosedextran 1 56 hydroxyethyl cellulose Ficoll 1 57 methyl cellulose Ficoll1 58 Zonyl poly(methacrylic acid) 1 59 Zonyl dextran 1 60 Zonylpolyacrylamide 1 61 Brij 35 poly(2-ethyl-2-oxazoline) 1 62 Brij 35Ficoll 1 63 Brij 35 polyallylamine 1 64 Tween 20 poly(methacrylic acid)1 65 Tween 20 poly(vinyl alcohol) 1 66 Tween 20poly(2-ethyl-2-oxazoline) 1 67 Tween 20 poly(ethylene glycol) 0 68 Tween20 dextran 0 69 Tween 20 Ficoll 1 70 Tween 20 polyacrylamide 1 71 Tween20 polyallylamine 1 72 Tween 20 hydroxyethyl cellulose 1 73 Triton X-100poly(methacrylic acid) 1 74 Triton X-100 poly(acrylic acid) 1 75 TritonX-100 poly(2-ethyl-2-oxazoline) 1 76 Triton X-100 dextran 0 77 TritonX-100 Ficoll 1 78 Triton X-100 polyacrylamide 1 79 Triton X-100polyallylamine 1 80 Triton X-100 hydroxyethyl cellulose 0 81 nonylphenolpolyoxyethylene (20) poly(methacrylic acid) 1 82 nonylphenolpolyoxyethylene (20) dextran 1 83 1-O-Octyl-B-D-glucopyranosidepoly(methacrylic acid) 1 84 1-O-Octyl-B-D-glucopyranosidepoly(2-ethyl-2-oxazoline) 1 85 1-O-Octyl-B-D-glucopyranosidepoly(ethylene glycol) 0 86 1-O-Octyl-B-D-glucopyranosidepolyethyleneimine 1 87 Pluronic F68 poly(methacrylic acid) 1 88 PluronicF68 poly(vinyl alcohol) 1 89 Pluronic F68 poly(2-ethyl-2-oxazoline) 1 90Pluronic F68 dextran 1 91 Pluronic F68 Ficoll 1 92 Pluronic F68polyacrylamide 1 93 Pluronic F68 polyethyleneimine 1 94 sodium dodecylsulfate poly(acrylic acid) 1 95 sodium cholate poly(methacrylic acid) 196 sodium cholate dextran sulfate 1 97 N,N-dimethyldodecylamine N-oxidepoly(methacrylic acid) 1 98 N,N-dimethyldodecylamine N-oxidepolyacrylamide 1 99 CHAPS poly(methacrylic acid) 1 100 CHAPSpoly(2-ethyl-2-oxazoline) 1 101 CHAPS poly(ethylene glycol) 1 102 CHAPSdextran 1 103 CHAPS Ficoll 1 104 CHAPS polyacrylamide 1 105 CHAPSpolyethyleneimine 1 106 CHAPS Pluronic F68 1 107 PVPNO PA 1 108 PVPNOPMAA 1 111 PVPNO PEOZ 1 112 PVPNO PEG 1 116 PVPNO PEI 1 117 PVPNO Tween1Multiphase Separation

Applicants used an identical approach to generate and optimize aqueousmultiphase polymer systems (AMPS) by combining three, four, or fivepolymer solutions into a single microcentrifuge tube, vortexing to mix,and accelerating layer segregation by centrifugation. Applicants reportthe four-phase and five-phase MPS found in this study in Tables S4 andS5, respectively.

As shown in Table S4 and S5, preliminary results suggest that there areseveral systems which did not result in phase separation, yet areinvolved in a verified four- or five-phase system. However, if acombination of multiple polymer aqueous phases results in aphase-separated aqueous polymer system, any sub-combination of themultiple polymer aqueous phases will also result in a phase-separatedaqueous polymer system. Accordingly, the systems that Applicants wereunable to produce as shown in Table S4 and S5 are in fact producibleusing routine optimization.

TABLE S3 List of identified three-phase systems: “0” refers to a MPSthat Applicant did not produce; “1” refers to a MPS produced. CountPolymers and Surfactants as phase components 1 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) 1 2 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) Ficoll 1 3 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) polyacrylamide 1 4 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(diallyldimethyl ammonium chloride 0 5poly(methacrylic acid) poly(2-ethyl-2-oxazoline) chondroitin sulfate A 16 poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyethyleneimine 1 7poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Tween 20 1 8poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Triton X-100 0 9poly(methacrylic acid) poly(2-ethyl-2-oxazoline)1-O-Octyl-B-D-glucopyranoside 1 10 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) Pluronic F68 1 11 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) CHAPS 1 12 poly(methacrylic acid)poly(ethylene glycol) Ficoll 1 13 poly(methacrylic acid) poly(ethyleneglycol) polyacrylamide 1 14 poly(methacrylic acid) poly(ethylene glycol)poly(diallyldimethyl ammonium chloride 0 15 poly(methacrylic acid)poly(ethylene glycol) polyethyleneimine 1 16 poly(methacrylic acid)poly(ethylene glycol) polyvinylpyrrolidone 1 17 poly(methacrylic acid)poly(ethylene glycol) Tween 20 1 18 poly(methacrylic acid) poly(ethyleneglycol) 1-O-Octyl-B-D-glucopyranoside 1 19 poly(methacrylic acid)poly(ethylene glycol) CHAPS 1 20 poly(methacrylic acid) Ficollpolyethyleneimine 1 21 poly(methacrylic acid) Ficoll Tween 20 1 22poly(methacrylic acid) Ficoll Triton X-100 1 23 poly(methacrylic acid)Ficoll Pluronic F68 1 24 poly(methacrylic acid) Ficoll CHAPS 1 25poly(methacrylic acid) polyacrylamide polyethyleneimine 1 26poly(methacrylic acid) polyacrylamide poly(propylene glycol) 1 27poly(methacrylic acid) polyacrylamide Zonyl 1 28 poly(methacrylic acid)polyacrylamide Tween 20 1 29 poly(methacrylic acid) polyacrylamideTriton X-100 1 30 poly(methacrylic acid) polyacrylamide Pluronic F68 131 poly(methacrylic acid) polyacrylamide N,N-dimethyldodecylamineN-oxide 1 32 poly(methacrylic acid) polyacrylamide CHAPS 1 33poly(methacrylic acid) polyethyleneimine carboxy-polyacrylamide 1 34poly(methacrylic acid) polyethyleneimine 1-O-Octyl-B-D-glucopyranoside 135 poly(methacrylic acid) polyethyleneimine Pluronic F68 1 36poly(methacrylic acid) polyethyleneimine CHAPS 1 37 poly(methacrylicacid) Pluronic F68 CHAPS 1 38 poly(acrylic acid) poly(ethylene glycol)polyacrylamide 1 39 poly(acrylic acid) poly(ethylene glycol)poly(diallyldimethyl ammonium chloride 0 40 poly(acrylic acid)polyacrylamide Triton X-100 0 41 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) 1 42 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) dextran 1 43 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) Ficoll 1 44 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) polyacrylamide 1 45 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) poly(diallyldimethyl ammonium chloride 0 46poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) dextran sulfate 1 47poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) chondroitin sulfate A 1 48poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) Tween 20 1 49 poly(vinylalcohol) poly(2-ethyl-2-oxazoline) Pluronic F68 1 50 poly(vinyl alcohol)poly(ethylene glycol) dextran 1 51 poly(vinyl alcohol) poly(ethyleneglycol) Ficoll 1 52 poly(vinyl alcohol) poly(ethylene glycol)polyacrylamide 1 53 poly(vinyl alcohol) poly(ethylene glycol)poly(diallyldimethyl ammonium chloride 0 54 poly(vinyl alcohol)poly(ethylene glycol) dextran sulfate 1 55 poly(vinyl alcohol)poly(ethylene glycol) Tween 20 1 56 poly(vinyl alcohol) dextran Ficoll 157 poly(vinyl alcohol) dextran Tween 20 1 58 poly(vinyl alcohol) dextranPluronic F68 1 59 poly(vinyl alcohol) Ficoll Tween 20 1 60 poly(vinylalcohol) Ficoll Pluronic F68 1 61 poly(vinyl alcohol) polyacrylamideTween 20 1 62 poly(vinyl alcohol) polyacrylamide Pluronic F68 1 63poly(2-ethyl-2-oxazoline) poly(ethylene glycol) dextran 1 64poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Ficoll 1 65poly(2-ethyl-2-oxazoline) poly(ethylene glycol) polyacrylamide 1 66poly(2-ethyl-2-oxazoline) poly(ethylene glycol) poly(diallyldimethylammonium chloride 0 67 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)dextran sulfate 0 68 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyethyleneimine 1 69 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)Tween 20 1 70 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)1-O-Octyl-B-D-glucopyranoside 1 71 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) CHAPS 1 72 poly(2-ethyl-2-oxazoline) dextranFicoll 1 73 poly(2-ethyl-2-oxazoline) dextran Tween 20 1 74poly(2-ethyl-2-oxazoline) dextran Triton X-100 1 75poly(2-ethyl-2-oxazoline) dextran Pluronic F68 1 76poly(2-ethyl-2-oxazoline) dextran CHAPS 1 77 poly(2-ethyl-2-oxazoline)Ficoll polyethyleneimine 1 78 poly(2-ethyl-2-oxazoline) Ficoll Brij 35 179 poly(2-ethyl-2-oxazoline) Ficoll Tween 20 1 80poly(2-ethyl-2-oxazoline) Ficoll Triton X-100 1 81poly(2-ethyl-2-oxazoline) Ficoll Pluronic F68 1 82poly(2-ethyl-2-oxazoline) Ficoll CHAPS 1 83 poly(2-ethyl-2-oxazoline)polyacrylamide polyethyleneimine 1 84 poly(2-ethyl-2-oxazoline)polyacrylamide Tween 20 1 85 poly(2-ethyl-2-oxazoline) polyacrylamideTriton X-100 1 86 poly(2-ethyl-2-oxazoline) polyacrylamide Pluronic F681 87 poly(2-ethyl-2-oxazoline) polyacrylamide CHAPS 1 88poly(2-ethyl-2-oxazoline) dextran sulfate poly(styrene sulfonic acid) 089 poly(2-ethyl-2-oxazoline) polyethyleneimine1-O-Octyl-B-D-glucopyranoside 1 90 poly(2-ethyl-2-oxazoline)polyethyleneimine Pluronic F68 1 91 poly(2-ethyl-2-oxazoline)polyethyleneimine CHAPS 1 92 poly(2-ethyl-2-oxazoline) Pluronic F68CHAPS 1 93 poly(ethylene glycol) dextran Ficoll 1 94 poly(ethyleneglycol) dextran polyvinylpyrrolidone 1 95 poly(ethylene glycol) dextranTween 20 1 96 poly(ethylene glycol) dextran CHAPS 1 97 poly(ethyleneglycol) Ficoll polyethyleneimine 1 98 poly(ethylene glycol) Ficoll Tween20 1 99 poly(ethylene glycol) Ficoll CHAPS 1 100 poly(ethylene glycol)polyacrylamide polyethyleneimine 1 101 poly(ethylene glycol)polyacrylamide Tween 20 1 102 poly(ethylene glycol) polyacrylamide CHAPS1 103 poly(ethylene glycol) polyethyleneimine1-O-Octyl-B-D-glucopyranoside 1 104 poly(ethylene glycol)polyethyleneimine CHAPS 1 105 dextran Ficoll hydroxyethyl cellulose 1106 dextran Ficoll Tween 20 1 107 dextran Ficoll Triton X-100 1 108dextran Ficoll Pluronic F68 1 109 dextran Ficoll CHAPS 1 110 dextranpolyvinylpyrrolidone poly(2-acrylamido-2-methyl-1-propanesulfonic acid)1 111 dextran hydroxyethyl cellulose Tween 20 1 112 dextran hydroxyethylcellulose Triton X-100 1 113 dextran Pluronic F68 CHAPS 1 114 Ficollpolyethyleneimine Pluronic F68 1 115 Ficoll polyethyleneimine CHAPS 1116 Ficoll hydroxyethyl cellulose Tween 20 1 117 Ficoll hydroxyethylcellulose Triton X-100 1 118 Ficoll Pluronic F68 CHAPS 1 119polyacrylamide polyethyleneimine Pluronic F68 1 120 polyacrylamidepolyethyleneimine CHAPS 1 121 polyacrylamide Pluronic F68 CHAPS 1 122polyethyleneimine Pluronic F68 CHAPS 1 123 PEOZ PEG PVPNO 0 124 PEOZ PEIPVPNO 0 125 PEOZ PA PVPNO 0 126 PEOZ PMAA PVPNO 0 127 PEG PEI PVPNO 0128 PEG PMAA PVPNO 0 129 PEG PA PVPNO 0 130 PEI PA PVPNO 0 131 PEI PMAAPVPNO 0 132 PA PMAA PVPNO 0 133 PEOZ PEG PVPNO 0 134 PEOZ TWEEN PVPNO 0135 PEOZ PA PVPNO 0 136 PEOZ PMAA PVPNO 0 137 PEG TWEEN PVPNO 0 138TWEEN PA PVPNO 0 139 TWEEN PMAA PVPNO 0 140 PA PMAA PVPNO 0 141 PEG PAPVPNO 0 142 PEG PMAA PVPNO 0

TABLE S4 List of identified four-phase systems: “0” refers to a MPS thatApplicant did not produce; “1” refers to a MPS produced. CONFIRMED? 0 -No, Count Phase components 1 - Yes 1 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Ficoll 1 2poly(methacrylic acid) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyacrylamide 1 3 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) poly(diallyldimethyl 0 ammonium chloride 4poly(methacrylic acid) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyethyleneimine 1 5 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) Tween 20 1 6 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) 1-O-Octyl-B-D- 0glucopyranoside 7 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) CHAPS 1 8 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) Ficoll polyethyleneimine 1 9 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) Ficoll Tween 20 1 10 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) Ficoll Triton X-100 0 11poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Ficoll Pluronic F68 112 poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Ficoll CHAPS 1 13poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyacrylamidepolyethyleneimine 1 14 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)polyacrylamide Tween 20 1 15 poly(methacrylic acid)poly(2-ethyl-2-oxazoline) polyacrylamide Triton X-100 1 16poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyacrylamide PluronicF68 1 17 poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyacrylamideCHAPS 1 18 poly(methacrylic acid) poly(2-ethyl-2-oxazoline)polyethyleneimine 1-O-Octyl-B-D- 0 glucopyranoside 1 19 poly(methacrylicacid) poly(2-ethyl-2-oxazoline) polyethyleneimine Pluronic F68 0 20poly(methacrylic acid) poly(2-ethyl-2-oxazoline) polyethyleneimine CHAPS1 21 poly(methacrylic acid) poly(2-ethyl-2-oxazoline) Pluronic F68 CHAPS1 22 poly(methacrylic acid) poly(ethylene glycol) Ficollpolyethyleneimine 1 23 poly(methacrylic acid) poly(ethylene glycol)Ficoll Tween 20 1 24 poly(methacrylic acid) poly(ethylene glycol) FicollCHAPS 1 25 poly(methacrylic acid) poly(ethylene glycol) polyacrylamidepolyethyleneimine 1 26 poly(methacrylic acid) poly(ethylene glycol)polyacrylamide Tween 20 1 27 poly(methacrylic acid) poly(ethyleneglycol) polyacrylamide CHAPS 1 28 poly(methacrylic acid) poly(ethyleneglycol) polyethyleneimine 1-O-Octyl-B-D- 0 glucopyranoside 1 29poly(methacrylic acid) poly(ethylene glycol) polyethyleneimine CHAPS 130 poly(methacrylic acid) Ficoll polyethyleneimine Pluronic F68 1 31poly(methacrylic acid) Ficoll polyethyleneimine CHAPS 1 32poly(methacrylic acid) Ficoll Pluronic F68 CHAPS 1 33 poly(methacrylicacid) polyacrylamide polyethyleneimine Pluronic F68 1 34poly(methacrylic acid) polyacrylamide polyethyleneimine CHAPS 1 35poly(methacrylic acid) polyacrylamide Pluronic F68 CHAPS 1 36poly(methacrylic acid) polyethyleneimine Pluronic F68 CHAPS 1 37poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) poly(ethylene glycol)dextran 1 38 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) Ficoll 1 39 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) polyacrylamide 1 40 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) poly(diallyldimethyl 0ammonium chloride 41 poly(vinyl alcohol) poly(2-ethyl-2-oxazoline)poly(ethylene glycol) dextran sulfate 1 42 poly(vinyl alcohol)poly(2-ethyl-2-oxazoline) poly(ethylene glycol) Tween 20 0 43 poly(vinylalcohol) poly(2-ethyl-2-oxazoline) dextran Ficoll 1 44 poly(vinylalcohol) poly(2-ethyl-2-oxazoline) dextran Tween 20 0 45 poly(vinylalcohol) poly(2-ethyl-2-oxazoline) dextran Pluronic F68 1 46 poly(vinylalcohol) poly(2-ethyl-2-oxazoline) Ficoll Tween 20 1 47 poly(vinylalcohol) poly(2-ethyl-2-oxazoline) Ficoll Pluronic F68 1 48 poly(vinylalcohol) poly(2-ethyl-2-oxazoline) polyacrylamide Tween 20 1 49poly(vinyl alcohol) poly(2-ethyl-2-oxazoline) polyacrylamide PluronicF68 1 50 poly(vinyl alcohol) poly(ethylene glycol) dextran Ficoll 1 51poly(vinyl alcohol) poly(ethylene glycol) dextran Tween 20 1 52poly(vinyl alcohol) poly(ethylene glycol) Ficoll Tween 20 1 53poly(vinyl alcohol) poly(ethylene glycol) polyacrylamide Tween 20 1 54poly(vinyl alcohol) dextran Ficoll Tween 20 1 55 poly(vinyl alcohol)dextran Ficoll Pluronic F68 1 56 poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) dextran Ficoll 1 57 poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) dextran Tween 20 1 58 poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) dextran CHAPS 1 59 poly(2-ethyl-2-oxazoline) poly(ethyleneglycol) Ficoll polyethyleneimine 0 60 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) Ficoll Tween 20 1 61 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) Ficoll CHAPS 1 62 poly(2-ethyl-2-oxazoline)poly(ethylene glycol) polyacrylamide polyethyleneimine 1 63poly(2-ethyl-2-oxazoline) poly(ethylene glycol) polyacrylamide Tween 201 64 poly(2-ethyl-2-oxazoline) poly(ethylene glycol) polyacrylamideCHAPS 1 65 poly(2-ethyl-2-oxazoline) poly(ethylene glycol)polyethyleneimine 1-O-Octyl-B-D- 1 glucopyranoside 66poly(2-ethyl-2-oxazoline) poly(ethylene glycol) polyethyleneimine CHAPS1 67 poly(2-ethyl-2-oxazoline) dextran Ficoll Tween 20 1 68poly(2-ethyl-2-oxazoline) dextran Ficoll Triton X-100 0 69poly(2-ethyl-2-oxazoline) dextran Ficoll Pluronic F68 1 70poly(2-ethyl-2-oxazoline) dextran Ficoll CHAPS 0 71poly(2-ethyl-2-oxazoline) dextran Pluronic F68 CHAPS 1 72poly(2-ethyl-2-oxazoline) Ficoll polyethyleneimine Pluronic F68 1 73poly(2-ethyl-2-oxazoline) Ficoll polyethyleneimine CHAPS 1 74poly(2-ethyl-2-oxazoline) Ficoll Pluronic F68 CHAPS 0 75poly(2-ethyl-2-oxazoline) polyacrylamide polyethyleneimine Pluronic F681 76 poly(2-ethyl-2-oxazoline) polyacrylamide polyethyleneimine CHAPS 177 poly(2-ethyl-2-oxazoline) polyacrylamide Pluronic F68 CHAPS 0 78poly(2-ethyl-2-oxazoline) polyethyleneimine Pluronic F68 CHAPS 1 79poly(ethylene glycol) dextran Ficoll Tween 20 1 80 poly(ethylene glycol)dextran Ficoll CHAPS 1 81 poly(ethylene glycol) Ficoll polyethyleneimineCHAPS 1 82 poly(ethylene glycol) polyacrylamide polyethyleneimine CHAPS1 83 dextran Ficoll hydroxyethyl cellulose Tween 20 1 84 dextran Ficollhydroxyethyl cellulose Triton X-100 1 85 dextran Ficoll Pluronic F68CHAPS 1 86 Ficoll polyethyleneimine Pluronic F68 CHAPS 1 87polyacrylamide polyethyleneimine Pluronic F68 CHAPS 1 88 PEOZ PEG PEIPVPNO 0 89 PEOZ PEG PA PVPNO 0 90 PEOZ PEI PA PVPNO 0 91 PEOZ PEI PMAAPVPNO 0 92 PEOZ PA PMAA PVPNO 0 93 PEG PEI PA PVPNO 0 94 PEG PEI PMAAPVPNO 0 95 PEG PA PMAA PVPNO 0 96 PEI PA PMAA PVPNO 0 97 PEOZ PEG PMAAPVPNO 0 98 PEOZ PEG PA PVPNO 0 99 PEOZ PEG TWEEN PVPNO 0 100 PEOZ TWEENPA PVPNO 0 101 PEOZ TWEEN PMAA PVPNO 0 102 PEOZ PA PMAA PVPNO 0 103 PEGTWEEN PA PVPNO 0 104 PEG TWEEN PMAA PVPNO 0 105 PEG PA PMAA PVPNO 0 106TWEEN PA PMAA PVPNO 0

TABLE S5 List of identified five-phase aqueous systems: “0” refers to aMPS that Applicant did not produce; “1” refers to a MPS produced.CONFIRMED? 0 - No, Count Polymers 1 - Yes 1 polyvinyl alcohol)poly(2-ethyl-2- poly(ethylene dextran Ficoll 1 oxazoline) glycol) 2poly(methacrylic poly(2-ethyl-2- poly(ethylene Ficoll polyethyleneimine1 acid) oxazoline) glycol) 3 poly(methacrylic poly(2-ethyl-2-poly(ethylene polyacrylamide polyethyleneimine 1 acid) oxazoline)glycol) 4 polyvinyl alcohol) poly(2-ethyl-2- poly(ethylene dextran Tween20 1 oxazoline) glycol) 5 poly(methacrylic poly(2-ethyl-2- poly(ethyleneFicoll Tween 20 1 acid) oxazoline) glycol) 6 polyvinyl alcohol)poly(2-ethyl-2- poly(ethylene Ficoll Tween 20 1 oxazoline) glycol) 7polyvinyl alcohol) poly(2-ethyl-2- dextran Ficoll Tween 20 1 oxazoline)8 polyvinyl alcohol) poly(ethylene dextran Ficoll Tween 20 1 glycol) 9poly(2-ethyl-2- poly(ethylene dextran Ficoll Tween 20 1 oxazoline)glycol) 10 poly(methacrylic poly(2-ethyl-2- poly(ethylene polyacrylamideTween 20 1 acid) oxazoline) glycol) 11 polyvinyl alcohol)poly(2-ethyl-2- poly(ethylene polyacrylamide Tween 20 1 oxazoline)glycol) 12 poly(methacrylic poly(2-ethyl-2- poly(ethylenepolyethyleneimine 1-O-Octyl-B-D- 0 acid) oxazoline) glycol)glucopyranoside 13 polyvinyl alcohol) poly(2-ethyl-2- dextran FicollPluronic F68 1 oxazoline) 14 poly(methacrylic poly(2-ethyl-2- Ficollpolyethyleneimine Pluronic F68 1 acid) oxazoline) 15 poly(methacrylicpoly(2-ethyl-2- polyacrylamide polyethyleneimine Pluronic F68 0 acid)oxazoline) 16 poly(methacrylic poly(2-ethyl-2- poly(ethylene FicollCHAPS 1 acid) oxazoline) glycol) 17 poly(2-ethyl-2- poly(ethylenedextran Ficoll CHAPS 1 oxazoline) glycol) 18 poly(methacrylicpoly(2-ethyl-2- poly(ethylene polyacrylamide CHAPS 1 acid) oxazoline)glycol) 19 poly(methacrylic poly(2-ethyl-2- poly(ethylenepolyethyleneimine CHAPS 1 acid) oxazoline) glycol) 20 poly(methacrylicpoly(2-ethyl-2- Ficoll polyethyleneimine CHAPS 1 acid) oxazoline) 21poly(methacrylic poly(ethylene Ficoll polyethyleneimine CHAPS 1 acid)glycol) 22 poly(2-ethyl-2- poly(ethylene Ficoll polyethyleneimine CHAPS1 oxazoline) glycol) 23 poly(methacrylic poly(2-ethyl-2- polyacrylamidepolyethyleneimine CHAPS 1 acid) oxazoline) 24 poly(methacrylicpoly(ethylene polyacrylamide polyethyleneimine CHAPS 1 acid) glycol) 25poly(2-ethyl-2- poly(ethylene polyacrylamide polyethyleneimine CHAPS 1oxazoline) glycol) 26 poly(methacrylic poly(2-ethyl-2- Ficoll PluronicF68 CHAPS 1 acid) oxazoline) 27 poly(2-ethyl-2- dextran Ficoll PluronicF68 CHAPS 1 oxazoline) 28 poly(methacrylic poly(2-ethyl-2-polyacrylamide Pluronic F68 CHAPS 1 acid) oxazoline) 29 poly(methacrylicpoly(2-ethyl-2- polyethyleneimine Pluronic F68 CHAPS 1 acid) oxazoline)30 poly(methacrylic Ficoll polyethyleneimine Pluronic F68 CHAPS 1 acid)31 poly(2-ethyl-2- Ficoll polyethyleneimine Pluronic F68 CHAPS 1oxazoline) 32 poly(methacrylic polyacrylamide polyethyleneimine PluronicF68 CHAPS 1 acid) 33 poly(2-ethyl-2- polyacrylamide polyethyleneiminePluronic F68 CHAPS 1 oxazoline) 34 PEG PEI PA PMAA PVPNO 0 35 PEOZ PEIPA PMAA PVPNO 0 36 PEOZ PEG PA PMAA PVPNO 0 37 PEOZ PEG PEI PMAA PVPNO 038 PEOZ PEG PEI PA PVPNO 0 39 PEOZ PEG PEI PA PMAA 0 40 PEG TWEEN PAPMAA PVPNO 0 41 PEOZ TWEEN PA PMAA PVPNO 0 42 PEOZ PEG TWEEN PMAA PVPNO0 43 PEOZ PEG TWEEN PA PVPNO 0 44 PEOZ PEG TWEEN PA PMAA 0

TABLE S6 List of six-phase aqueous MPS: “0” refers to a MPS thatApplicant did not produce; “1” refers to a MPS produced. Count Phasecomponents 1 poly(vinyl poly(2-ethyl- poly(ethylene dextran′ Ficoll′Tween 20′ 1 alcohol)′ 2-oxazoline)′ glycol)′ 2 PMAA PEOZ PEG Ficoll PEICHAPS 0 3 PMAA PEOZ PEG PA PEI CHAPS 1 4 PMAA PEOZ PEI Ficoll CHAPS P680 5 PMAA PEOZ PA PEI P68 CHAPS 1

Poly(2-vinylpyridine-N-oxide) Multiphase Systems

Abbreviations for polymer used in this study:

-   poly(2-vinylpyridine-N-oxide)-PVPNO-   poly(methacrylic acid)-PMAA-   poly(acrylic acid)-PAA-   polyacrylamide-PA-   poly(vinyl alcohol)-PVA-   poly(2-ethyl-2-oxazoline)-PEOZ-   poly(ethylene glycol)-PEG-   hydroxyethylcelluolose-HEC-   polyethyleneimine-PEI-   polyvinylpyrrolidone-PVP

Indicators for phase-separation results:

-   −miscible-   +immiscible-   x incompatible

The phase separation results obtained are shown below:

PVPNO-PA (+)

PVPNO-PMAA (+)

PVPNO-PAA (x)

PVPNO-PVA (−)

PVPNO-PEOZ (+)

PVPNO-PEG (+)

PVPNO-HEC (−)

PVPNO-Dextran (−)

PVPNO-Ficoll (−)

PVPNO-PEI (+)

PVPNO-Tween (+)

PVPNO-PVP (−)

Two 6-Phase systems are contemplated:

PEOZ-PEG-PEI-PA-PMAA-PVPNO; and

PEOZ-PEG-TWEEN-PA-PMAA-PVPNO.

The following three-phase systems are contemplated: PEOZ-PEG-PVPNO,PEOZ-PEI-PVPNO, PEOZ-PA-PVPNO, PEOZ-PMAA-PVPNO, PEG-PEI-PVPNO,PEG-PMAA-PVPNO, PEG-PA-PVPNO, PEI-PA-PVPNO, PEI-PMAA-PVPNO,PA-PMAA-PVPNO, PEOZ-PEG-PVPNO, PEOZ-TWEEN-PVPNO, PEOZ-PA-PVPNO,PEOZ-PMAA-PVPNO, PEG-TWEEN-PVPNO, TWEEN-PA-PVPNO, TWEEN-PMAA-PVPNO,PA-PMAA-PVPNO, PEG-PA-PVPNO, and PEG-PMAA-PVPNO.

The following four-phase systems are contemplated: PEOZ-PEG-PEI-PVPNO,PEOZ-PEG-PA-PVPNO, PEOZ-PEI-PA-PVPNO, PEOZ-PEI-PMAA-PVPNO,PEOZ-PA-PMAA-PVPNO, PEG-PEI-PA-PVPNO, PEG-PEI-PMAA-PVPNO,PEG-PA-PMAA-PVPNO, PEI-PA-PMAA-PVPNO, PEOZ-PEG-PMAA-PVPNO,PEOZ-PEG-PA-PVPNO, PEOZ-PEG-TWEEN-PVPNO, PEOZ-TWEEN-PA-PVPNO,PEOZ-TWEEN-PMAA-PVPNO, PEOZ-PA-PMAA-PVPNO, PEG-TWEEN-PA-PVPNO,PEG-TWEEN-PMAA-PVPNO, PEG-PA-PMAA-PVPNO, and TWEEN-PA-PMAA-PVPNO (seeExperimental Section for the acronym of the polymers).

The following five-phase systems are contemplated:PEG-PEI-PA-PMAA-PVPNO, PEOZ-PEI-PA-PMAA-PVPNO, PEOZ-PEG-PA-PMAA-PVPNO,PEOZ-PEG-PEI-PMAA-PVPNO, PEOZ-PEG-PEI-PA-PVPNO, PEOZ-PEG-PEI-PA-PMAA,PEG-TWEEN-PA-PMAA-PVPNO, PEOZ-TWEEN-PA-PMAA-PVPNO,PEOZ-PEG-TWEEN-PMAA-PVPNO, PEOZ-PEG-TWEEN-PA-PVPNO, andPEOZ-PEG-TWEEN-PA-PMAA (see Experimental Section for the acronym of thepolymers).

Multiphase Systems of Liquid Polymers

The following liquid polymers were used for preparing solvent-free MPScomprising liquid polymer phases.

(1)poly(propylene glycol)-PPG; INSOLUBLE in water.

(2) poly(ethylene glycol)-PEG; molecular weight <1000 Da (above thismolecular weight, it is a solid); SOLUBLE in water.

(3) Pluronic L121-PL, a PEG-PPG block co-polymer is a liquid; INSOLUBLEin water

(4) polydimethylsiloxane-PDMS; INSOLUBLE in water.

(5) poly(ethyl vinyl ether)-PEVE; INSOLUBLE in water

(6) polybutadiene-PBD; INSOLUBLE in water

The following 2 phases liquid polymer MPSs were prepared:

PPG-PEG;

PPG-PL;

PPG-PDMS;

PPG-PBD;

PEG-PL;

PEG-PDMS;

PEG-PEVE;

PEG-PBD;

PL-PDMS;

PL-PBD;

PDMS-PEVE;

PDMS-PBD; and

PEVE-PBD.

The following 3 phases liquid polymer MPSs were studied: (0=predicted;1=confirmed by preparation)

PPG-PEG-PL (1);

PPG-PDMS-PBD (1);

PPG-PEG-PBD (1);

PEG-PEVE-PBD (1);

PEG-PEVE-PDMS (1);

PPG-PEG-PDMS (1);

PEG-PL-PDMS (0);

PEG-PL-PBD (1);

PL-PDMS-PBD (1); and

PDMS-PEVE-PBD (1).

The following 4 phases liquid polymer MPSs were studied: (0=predicted;1=confirmed by preparation)

PPG-PEG-PL-PDMS (0);

PPG-PEG-PL-PBD (0);

PPG-PEG-PDMS-PBD (1);

PPG-PL-PDMS-PBD (0); and

PEG-PL-PDMS-PBD (1).

The following 4 phases liquid polymer MPS was predicted:

PPG-PEG-PL-PDMS-PBD (0).

Example 2. Preparation of a Model MPS Separations System

Applicants used immiscible aqueous polymer solutions of polyvinylalcohol (PVA), PEG, and dextran to form three discontinuous densitybarriers with designed densities for partitioning different analyteswith different densities.

Applicants have previously reported on the utilization of an eggbeateras a portable centrifuge for point-of-care diagnostics (softcentrifuge). Briefly, a length of heat-sealed polyethylene tubing thatcontains the sample is taped to a blade of an eggbeater paddle. Manualcranking of the eggbeater can comfortably achieve relative centrifugalforces (RCF) ranging from 240-350×g.

Applicants prepared the triphasic aqueous density barriers by firstmixing each polymer and then pipetting the mixture into the tubing. Thethree polymer phases spontaneously formed after five minutes ofcentrifugation using the eggbeater. Applicants then removed the tubingfrom the blade, introduced 5-10 μL of sample to the top layer (by thedisplacement of a thin wire), and reapplied the tubing to the paddle.

Applicants used individual solutions of polymers to demonstrate thesensitive (Δρ≥0.001 g/cm³) and rapid separation of microsphere standardsacross the density barrier. For example, the density of a 15 wt %solution of poly(styrene sulfonic acid) (PSSA) 500 k is 1.062 g/cm³ anda green-dyed microsphere standard with a density of 1.063 g/cm³ pelletsin less than five minutes (FIG. 3(A)). Conversely, this same standardwill stack on top of a 40 wt % solution of PEG 20 k (ρ=1.067 g/cm³; FIG.3(B)).

Example 3. AMPS Comprising Surfactants

Properties of the surfactants used are shown in Table S7.

TABLE S7 Properties of the surfactants Applicants have used in thisstudy. Surfactant Mw (g/mol) Concentration Density g/cm³ Non-Ionic AZonyl N/A 50% (v/v) 1.037 B Brij 35 ~1,198 30% (v/v) 1.025 C Tween 20~1,228 45% (v/v) 1.067 D Triton X-100 ~625 20% (v/v) 1.017 E Nonylphenolpolyoxyethylene (20) ~13,420 40% (v/v) 1.035 F1-O-Octyl-β-D-glucopyranoside 292.37 100 mg/ml 1.011 G Pluronic F68~8,400 340 mg/ml 1.049 Ionic (negative) H sodium dodecylsulfate 288.38350 mg/ml 0.998 I DOWFAX 2A1 N/A 67% (v/v) 1.096 J sodium cholate 408.57430 mg/ml 0.997 Zwitterionic K N,N-dimethyldodecylamine N-oxide 229.424% (v/v) 1.123 L CHAPS 614.88 250 mg/ml 1.042Phase Separation

Applicants added equal volumes of polymer and surfactant solutions (200μL) into an Eppendorf tube (plastic test tube), thoroughly mixed thecontents through vortexing for at least 30 seconds, and centrifuge thetube at 2,000×g for at least 10 minutes.

Coloring of Multiphase Systems

Applicants added the components of a multiphase system in equal volumesalong with 10 uL of a 10 mg/ml solution of a desired dye such as allurared or ethyl orange into an Eppendorf tube and thoroughly mixed thecontents through vortexing for at least 30 seconds. Thereafter,Applicants transferred this homogenous solution into a sealed capillaryglass tube with a syringe, and centrifuged this capillary tube at6,000×g for 30 minutes. Lists of 2-, 3-, and 4-phase systems preparedwith polymers and surfactants are found in Tables S2, S3 and S4,respectively.

Example 4. MPS in Patterned Paper

FIG. 10 (A) illustrates a phase-separated poly(ethyleneglycol)/poly(methacrylic acid) (PEG/PMAA) two-phase system (left) andphase-separated PEG/PMAA/Allura Red system (right), wherein Allura Redselectively stained the top phase. As shown in FIG. 10 (B), Applicantsseparated the top and bottom phases and spotted them onto a wax-printedpaper. The paper was folded such that the phases came into contact. Evenafter close contact, Applicants did not observe the intermixing of thephases, as demonstrated by the fact that the Allura Dye remained in thehydrophilic region 1.

In another experiment, selective diffusion of Allura Red in PEG/PMAAtwo-phase system on paper is shown (FIG. 11). As shown in FIG. 11(A)(i),20 mg/ml Allura Red solution was loaded on the hydrophilic regions onwax-printed paper and dried. As shown in FIG. 11(A)(ii), the top andbottom phases of a PEG/PMAA two-phase system were spotted onto anotherwax-printed paper, the paper was folded and sandwiched the dried AlluraRed-spotted paper in between the top of bottom phases through folding.The solution in the top and bottom phases and the Allura Red wereallowed to be in contact and in equilibrium for about 5 minutes and thenthe paper was unfolded. As shown in FIG. 11B, the dissolved dyepredominantly accumulated in the top phases.

Example 5. MPS in Organic Solvent

Applicants have discovered that certain polymers and surfactant willphase-separate in organic solution. A summary of polymers andsurfactants used in organic MPS is listed in Table S12. Tables S12 showsthe properties of the polymers stock solutions in dichloromethane:weight average molecular weights of the polymers (kDa), concentrations(% w/v), densities (g/cm³), and refractive indices.

TABLE S12 List of polymers, their acronym and surfactants used inorganic MPS. avg. Mw density refractive polymer acronym (kDa) % w/v(g/cm³) index 1 poly(bisphenol A carbonate) PBPA 64 30 1.484 2Polysulfone PSulf 67 15 1.455 3 Polydimethylsiloxane 200 ® PDMS   1.3 501.409 4 Polystyrene PS 50 30 1.480 5 poly(4-vinylpyridine) PVPy 50 301.298 1.492 6 Polyvinylpyrrolidone PVP 55 35 1.482 7poly(2-ethyl-2-oxazoline) PEOZ 50 30 1.284 1.474 8 Polyethyleneimine PEI25 30 1.461 9 Poly(methylmethacrylate-co-methacrylic PMAA-PMMA 34 301.447 acid) (1:0.016) copolymer 10 poly(methyl methacrylate) PMMA 35 301.299 1.461 11 polyoxyethylene (20) sorbitan monolaurate Tween   1.2 501.240 1.450 12 poly(tetrahydrofuran) PTHF   2.9 30 1.442 13poly(propylene glycol) PPG  4 35 1.439 14 poly(ethylene glycol)PEG_(20k) 20 40 1.454 15 polyvinyl acetate) PVA 100  30 1.436 16Polycaprolactone PCL  80* 25 1.447 17 poly(ethylene glycol) PEG_(100k)100  20 18 poly(ethylene glycol) monomethyl ether PEG_(2k)  2 40 19Triethylene glycol monomethyl ether TEG    0.16 40 20 Poly(methacrylicacid) PMAA 34 30 1.447 *Number average molecular weight.

A six-phase organic MPS was produced in this study. The MPS was producedusing dichloromethane as the solvent and the phase component compositionof the six phases are PBPA-PDMS-PS-PVPy-PEOZ-PCL.

Also, various two-phase organic MPS were investigated and the result issummarized in FIG. 5. As shown in FIG. 5,dichloromethane-polymer-polymer ternary mixtures were categorized aseither biphasic (+), homogeneous (−), or incompatible (0). 76 mixtures(63%) formed two phases, 39 mixtures are homogeneous (33%), and 5mixtures (4%) are incompatible. Asterisks indicated mixtures that wereformed at half the concentration of the stock polymers.

Example 6. MPS Comprising Aqueous and Non-Aqueous Phases

A 10-phase MPS is shown in FIG. 12. The MPS was constructed by mixingmineral oil, ionic liquid, silicone oil, perfluorohexane, mercury, andfive aqueous phase component stock solutions (FIG. 12A). After themixture was shaken thoroughly, it was allowed to phase separate to formthe 10-phase MPS as shown in FIG. 12B. From the top to bottom, the10-phase MPS has the following phases: a mineral oil phase, a ionicliquid phase, a silicone oil phase, 5 aqueous phases, a perfluorohexanephase, and a mercury phase. The 10 phases are arranged according todensity.

Density Calculation for Beads

Polymers and Beads. Applicants purchased the following polymers:poly(ethylene glycol) (Sigma-Aldrich; MW=20,000 Da), Ficoll (Alfa Aesar;MW=400,000 Da), and dextran (Spectrum Chemical; 250,000 Da). Applicantsused each polymer without further purification, and prepared stockaqueous solutions at a concentration of 30% (wt/vol) without adding saltor titrating the pH. Applicants purchased a series of glass densitystandard floats (i.e., “beads”) from American Density Materials, thedensities of which spanned 1.0400 g/cm³ to 1.1000 g/cm³. The vendorcertified that the density of each bead was calibrated to +0.0002 g/cm³at 23° C., and the average diameter of each bead was approximately 5 mm.Applicants purchased polystyrene beads of uncharacterized density fromMcMaster-Carr. The average diameter of these beads was ⅛″ (3.175 mm).

Phase Separation and Analysis.

To prepare aqueous two-phase polymer systems (ATPS), Applicants addedequivalent volumes of polymer solutions (either at stock concentrationsor a dilution) into a container (e.g., Eppendorf tube or plasticcuvette), thoroughly mixed the solutions by vortex for 30 seconds, andaccelerated phase separation by centrifugation at 2000 g for 2-10minutes. Phase separation would occur in the absence of centrifugation(i.e., under a standard gravitational field of Ig) over a period of timethat depended on the viscosity of the solutions and the difference indensity between each phase, anywhere from minutes to hours. Applicantsremoved an aliquot of each phase (ca. 800 μL) in order to analyze thedensity of each layer by oscillating U-tube densitometry (Anton PaarDM35N). Applicants measured the interfacial tension between the top andbottom phases by the spinning drop method using a tensiometer.

Separation of Beads Based on Density.

Using the density step produced by a dextran-Ficoll ATPS, Applicantsdemonstrated in the manuscript that each two-phase system could separateobjects based on density into three positions: (i) floating at thesurface of the top phase, (ii) at the interface between phases, and(iii) settled at the bottom of the container. This type of separationcan occur independently of the densities of each aqueous phase, themagnitude of the density step, and the polymers employed to generate theATPS. In FIG. 8, Applicants demonstrate the separation of three beadsusing the density step produced by a poly(ethylene glycol)-Ficoll ATPS(Δρ=0.0802 g/cm³; ρ_(t)=1.0320 g/cm³; ρ_(b)=1.1122 g/cm³).

Using the Interface to Calculate Densities.

It was observed that the absolute position of a bead captured at theinterface between layers of an ATPS depended on the density of the bead,the densities of each layer, and the contact angle between the bead andthe interface. See equation (1). Eq. 1 can be expressed as a function ofdensities and several geometric parameters (i.e., angles and distances),as depicted in FIG. 9.

Applicants are given a sphere with a radius R (m) and density ρ₀ (g/cm³)sitting at equilibrium at an interface between two liquids. The topliquid has a density p, (g/cm³), the bottom liquid has a density ρ_(b)(g/cm³). The level between the liquids at equilibrium is at a height ofC (m) in the z direction,C=−R cos α  (2)where α is the inclination angle (deg) measured from the negative z axisup towards the origin. Similarly defined, the angle ϕ (deg) indicateswhere the sphere is incident with the interface. Let θ_(c) (deg) definethe contact angle between the surface of the sphere and the interface;hence, θ_(c)+ϕ−π (deg) is the inclination angle of the liquid/liquidinterfacial tension (γ_(br); N/m) in the z direction.

Applicants can simplify the calculations by shifting to the relativebouyant densities; Applicants define ρ_(R)=ρ_(b)−ρ_(t) andρ_(S)=ρ₀−ρ_(t).

The Gravitational Force, F_(g)

$\begin{matrix}{F_{g} = {{- \frac{4}{3}}\pi\; R^{3}\rho_{S}g}} & (3)\end{matrix}$

The Buoyant Force, F_(B), Derived from the Hydrostatic PressureIntegral.

The hydrostatic pressure is only a function of the depth of the beadinto the bottom layer, in this case:P(θ)=ρ_(R) g(C−R cos θ){circumflex over (z)}  (4)

Where C is defined from above to give us F (α)=QF _(B) =

P·{circumflex over (n)}dS  (5)F _(B)=2πρ_(R) g∫ _(π−ϕ) ^(π)(C−R cos θ)R ² sin θ Cos θdθ  (6)F _(B)=2πρ_(R) g∫ _(π−ϕ) ^(π) CR ² sin θ cos θdθ−2πρ_(R) g∫ _(π−ϕ) ^(π)R cos θR ² sin θ cos θdθ  (7)F _(B)=πρ_(R) gCR ²∫_(π−ϕ) ^(π)2 sin θ cos θdθ+2πR ³ρ_(R) g∫ _(π−ϕ) ^(π)cos² θdθ  (8)Note that 2 sin θ cos θ=sin(2θ) and let θ′=2θ.

$\begin{matrix}{\mspace{79mu}{F_{B} = {{\frac{1}{2}{\pi\rho}_{R}{gCR}^{2}{\int_{{2\pi} - {2\phi}}^{2\pi}{\sin\;\theta^{\prime}d\;\theta^{\prime}}}} + {2\pi\; R^{3}\rho_{R}{g\left( \frac{\cos^{3}\theta}{3} \right)}}}}}_{\pi}^{\pi - \phi} & (9) \\{F_{B} = {{\frac{1}{2}{\pi\rho}_{R}{{gCR}\left( {{- {\cos\left( {2\pi} \right)}} + {\cos\left( {{2\pi} - {2\phi}} \right)}} \right)}} + {\frac{2}{3}\pi\; R^{3}{\rho_{R}\left( {{\cos^{3}\left( {\pi - \phi} \right)} - {\cos^{3}\pi}} \right)}}}} & (10) \\{\mspace{79mu}{F_{B} = {{\frac{1}{2}{\pi\rho}_{R}{{gCR}^{2}\left( {{- 2}\sin^{2}\phi} \right)}} + {\frac{2}{3}{\pi\rho}_{R}{{gR}^{3}\left( {{{- \cos^{3}}\phi} + 1} \right)}}}}} & (11) \\{\mspace{79mu}{F_{B} = {{{- {\pi\rho}_{R}}{{gCR}^{2}\left( {\sin^{2}\phi} \right)}} + {\frac{2}{3}{\pi\rho}_{R}{{gR}^{3}\left( {1 - {\cos^{3}\phi}} \right)}}}}} & (12)\end{matrix}$If the distance between the equilibrium level and the wetted surface isd (m),C+d=R cos ϕ

C=R cos ϕ−d  (13)

$\begin{matrix}{F_{B} = {{{- {\pi\rho}_{R}}{{gR}^{3}\left( {{\cos\;\phi} - {\cos^{3}\phi}} \right)}} + {{\pi\rho}_{R}{gdR}^{2}\sin^{2}\phi} + {\frac{2}{3}{\pi\rho}_{R}{gR}^{3}} - {\frac{2}{3}{\pi\rho}_{R}{gR}^{3}\cos^{3}\phi}}} & (14) \\{F_{B} = {{{- {\pi\rho}_{R}}{gR}^{3}\cos\;\phi} + {{\pi\rho}_{R}{gR}^{3}\cos^{3}\phi} + {{\pi\rho}_{R}{gdR}^{2}\sin^{2}\phi} + {\frac{2}{3}{\pi\rho}_{R}{gR}^{3}} - {\frac{2}{3}{\pi\rho}_{R}{gR}^{3}\cos^{3}\phi}}} & (15) \\{F_{B} = {{{- {\pi\rho}_{R}}{gR}^{3}\cos\;\phi} + {{\pi\rho}_{R}{gdR}^{2}\sin^{2}\phi} + {\frac{2}{3}{\pi\rho}_{R}{gR}^{3}} + {\frac{1}{3}{\pi\rho}_{R}{gR}^{3}\cos^{3}\phi}}} & (16)\end{matrix}$

The Interfacial Tension, F_(I).

Exploiting the rotational symmetry, Applicants just need the z componentof the interfacial tension, γ_(bt), times the line over which it isapplied:F _(I) =L×γ _(bt) ^(z)  (17)F _(I)=2πRγ _(bt) sin ϕ cos(θ_(c)+ϕ−π)  (18)

Equation for Density of an Object.

Applicants can now substitute eq. 3, eq. 16, and eq. 18 into eq. 1, andset their sum to zero:

$\begin{matrix}{0 = {{{- \frac{4}{3}}{\pi\rho}_{S}{gR}^{3}} - {{\pi\rho}_{R}{gR}^{3}\cos\;\phi} + {{\pi\rho}_{R}{gdR}^{2}\sin^{2}\phi} + {\frac{2}{3}{\pi\rho}_{R}{gR}^{3}} + {\frac{1}{3}{\pi\rho}_{R}{gR}^{3}\cos^{3}\phi} + {2\pi\; R\;\gamma_{bt}\sin\;{{\phi cos}\left( {\theta_{c} + \phi - \pi} \right)}}}} & (19) \\{0 = {{- \rho_{S}} - {\frac{3}{4}\rho_{R}\cos\;\phi} + {\frac{3}{4}\rho_{R}\frac{d}{R}\sin^{2}\phi\frac{1}{2}\rho_{R}} + {\frac{1}{4}\rho_{R}\cos^{3}\phi} + {\frac{3\gamma_{bt}}{2{gR}^{2}}\sin\;{{\phi cos}\left( {\theta_{c} + \phi - \pi} \right)}}}} & (20)\end{matrix}$

Applicants then substitute the original densities for ρ_(R) and ρ_(S):

$\begin{matrix}{0 = {{- \left( {\rho_{1} - \rho_{3}} \right)} - {\frac{3}{4}\left( {\rho_{2} - \rho_{3}} \right)\cos\;\phi} + {\frac{3}{4}\left( {\rho_{2} - \rho_{3}} \right)\frac{d}{R}\sin^{2}\phi} + {\frac{1}{2}\left( {\rho_{2} - \rho_{3}} \right)} + {\frac{1}{4}\left( {\rho_{2} - \rho_{3}} \right)\cos^{3}\phi} + {\frac{3\gamma_{bt}}{2{gR}^{2}}\sin\;{{\phi cos}\left( {\theta_{c} + \phi - \pi} \right)}}}} & (21)\end{matrix}$

Applicants can now solve for the unknown density, ρ₀, of the bead:

$\begin{matrix}{\rho_{0} = {\frac{\left( {\rho_{b} + \rho_{t}} \right)}{2} + {\frac{\left( {\rho_{b} - \rho_{t}} \right)}{4}\left\lfloor {{\cos^{3}\phi} - {3\cos\;\phi} + {3\frac{d}{R}\sin^{2}\phi}} \right\rfloor} + {\frac{3\gamma_{bt}}{2{gR}^{2}}\sin\;{{\phi cos}\left( {\theta_{c} + \phi - \pi} \right)}}}} & (22)\end{matrix}$

Geometric Measurements.

Applicants use a camera to acquire images of the beads captured at theinterface. Applicants import the images into Adobe Illustrator in orderto (i) outline the border of the bead, (ii) determine the position ofthe center of the bead, and (iii) identify the point of intersectionbetween the bead and the interface. Using these guides, Applicantsmeasure R, d, ϕ, and θ_(c) using ImageJ or othe similar imageediting/analysis software such as MATLAB, Gimp, etc. ImageJ measuresangle absolutely, but all distances are measured in pixels. Applicantsuse the standardized width of the cuvette (10 mm) to calibrate z and Rmeasurements.

A PEG-Ficoll ATPS with a large density step (Δρ=0.0802 g/cm³) wasprepared to analyze several density standard beads over a range ofdensities using a single interface using equation (2) (Table S12).Geometric measurements routinely calculated the density of a bead to anaccuracy better than 1% over densities that spanned 1.0400 g/cm³ to1.1000 g/cm³ using this density step. This method was used to determinethe unknown density of a polystyrene bead. Using the geometricmeasurements of the bead at the interface, the density of thepolystyrene bead was calculated to be 1.0466 g/cm³, which was inexcellent agreement to its density as measured by magnetic levitation(1.0456 g/cm³). Since the interfacial tension, γ_(bt), is very small (onthe order of μN/m, typically) for ATPS, the buoyancy correction termdominates equation 2. Therefore, smaller density steps should providemore accurate density measurements. Using the density step generated bythe dextran-Ficoll ATPS (Δρ=0.0023 g/cm³), the density of a glass beadcaptured at the interface was measured to a very high precision, 0.06%or 0.0006 g/cm³.

TABLE S12 Comparison of densities measured using ATPS. For a set ofbeads, Applicants compare the known densities to those measuredgeometrically using the density step between layers and the position ofthe beads at the ATPS interface. Density Density Density Step KnownDensity^(†) Measured Density^(‡) Difference Difference Material (g/cm³)(g/cm³) (g/cm³) (g/cm³) (%) 0.080 glass 1.0400 ± 0.0002 1.0367 −0.0033−0.32 polystyrene 1.0456 ± 0.0009 1.0466 0.0010 0.10 glass 1.0500 ±0.0002 1.0489 −0.0011 −0.10 glass 1.0600 ± 0.0002 1.0464 −0.0136 −1.28glass 1.0700 ± 0.0002 1.0616 −0.0084 −0.79 glass 1.0800 ± 0.0002 1.0689−0.0122 −1.02 glass 1.0900 ± 0.0002 1.0812 −0.0088 −0.81 glass 1.1000 ±0.0002 1.0924 −0.0076 −0.68 0.002 glass 1.0790 ± 0.0002 1.0796 0.00060.06 ^(†)Each glass bead's density and density tolerances is certifiedby vendor. The density of polystyrene beads is culcualted. The standarddeviation was calculated from seven measurements. ^(‡)The averagedensity and standard deviation for each bead were calculated from sevenmeasurements.

Example 6. An Aqueous MPS Deposited on Paper

As shown by FIG. 10A, a two-phase aqueous MPS using poly(ethyleneglycol) (PEG) and poly(methacrylic acid) (PMAA) as the phase componentin each phase is shown to form in a tube (FIG. 10A). Also shown in FIG.10A, Allura Red, a chemical able to selectively accumulate in the PEGphase, is included to help the visualization of the phase separation. InFIG. 10B, a patterned paper is shown where hydrophic regions, e.g.,hydrophilic regions 1 and 2 (light-gray circle), are surrounded byhydrophobic barrier (black regions). The top phase containing PEG andAllura Red is loaded onto hydrophilic region 1 and the bottom phase isloaded onto hydrophilic region 2 (FIG. 10B(i)). As shown in FIG. 10B,the patterned paper is then folded along the line shown so thathydrophilic regions 1 and 2 are allowed to be in contact and the twophases are in equilibrium (FIG. 10B(ii)). The two phases remain in phaseseparation as demonstrated by the fact that the Allura Red dye, shown bythe dark gray color, remains in the hydrophilic region 1 only.

Example 7. Shifting the Density Range of a MPS Using D₂O

Applicants prepared stock solutions of 20% (wt/vol)poly(2-ethyl-2-oxazoline) and 30% (wt/vol) poly(ethylene glycol) inaqueous solutions whose water content ranged from 100% H₂O to 70%D₂O/30% H₂O. Applicants generated a series of two-phase AMS frommixtures of these solutions and measured the densities of each phaseusing densitometry (FIG. 13).

The relationship between the concentration of D₂O added to the mixtureof polymer solutions and the density of each phase is linear (R²>0.998),but the slopes of the linear fit to each set of data are not parallel:the slopes, m (in units of g/cm³/% D₂O), for the top and bottom phasesare 8.9E-4 and 7.4E-4, respectively.

These results do not provide evidence that D₂O partitions between thephases of the AMS; rather, these results are consistent with theobservation that the concentrations of solutes differ between phases ofaqueous two-phase systems. Since a greater concentration of solutes isrelated to a greater occupied solution mass fraction, the phase withless total water will incorporate less total D₂O at equivalentconcentrations. The density of this phase will thus increase at lowerrate compared to the density of a phase with a lower concentration ofsolutes (i.e., more water).

Applicants used ¹H-NMR to quantify the concentration of water in eachphase: the concentrations of water in the top and bottom phases were38.5 M (69% by volume) and 31.8 M (57% by volume), respectively. Theratio of the concentrations of water in each phase (1.2) is equivalentto the ratio of the slopes that we observe for the phase density as afunction of D₂O concentration.

Example 8. Shifting the Density Range of a MPS Using Salts

Applicants also examined the use of adding water-soluble salts into AMSas a method to increase the densities of the phases of an AMSindependently of the magnitude of the step in density between phases.

Applicants prepared a series of three-phase AMS from mixtures of 30%(wt/vol) poly(ethylene glycol), 40% (wt/vol) Ficoll, and 40% (wt/vol)poly(methacrylic acid) that also included alkali bromide salts, one oflithium bromide through cesium bromide, at concentrations from 0 M to 2M. Although these salts are soluble in water at concentrations greaterthan 5 M, the presence of polymers in solution lowered the solubility ofthe salts. After phase separation, we removed samples from each phaseand measured their densities using densitometry. FIG. 14 illustrates theeffects on phase density after LiBr and CsBr salts were added to theMPSs. In FIG. 14, the data points are 0 M salt concentration correspondsto a MPS without any salt added. The densities of the phases increasedwith the increasing salt concentrations.

Upon review of the description and embodiments of the present invention,those skilled in the art will understand that modifications andequivalent substitutions may be performed in carrying out the inventionwithout departing from the essence of the invention. Thus, the inventionis not meant to be limiting by the embodiments described explicitlyabove, and is limited only by the claims which follow.

The invention claimed is:
 1. A method of analyzing or separating asample comprising: providing a phase-separated system comprising atleast two phases, wherein a) the at least two phases each comprises aphase component selected from the group consisting of a polymer, asurfactant and combinations thereof, wherein at least one phasecomprises a polymer; each said phase has an upper and a lower phaseboundary; and each of the two or more phases has a different density andthe phases, taken together, represent a density gradient; and b)introducing a sample comprising one or more analytes of interest to thephase-separated system without disrupting the phase boundaries of thephase-separated solution; and c) allowing each of the analytes tomigrate to a location in the phase-separated system that ischaracteristic of its density, wherein during migration the samplecontacts one or more of the two or more phases sequentially; wherein thephase-separated system is supported along a filament or on a sheet. 2.The method of claim 1, wherein greater than 80% of the analyte islocated at one or more of the phase boundaries.
 3. The method of claim1, wherein greater than 90% of the analyte is located at one or more ofthe phase boundaries.
 4. The method of claim 1, wherein the samplecomprises a plurality of analytes and each analyte migrates to adifferent location in the phase-separated system.
 5. The method of claim1, wherein after migration, the analyte resides at a boundary location.6. The method of claim 1, wherein the boundary location is at aninterface between a phase with a density greater than the density of theanalyte and a phase with a density that is less than the density of theanalyte.
 7. The method of claim 1, wherein after migration, the analyteresides within a phase of the phase-separated system whose densitymatches the density of the analyte.
 8. The method of claim 1, whereinthe phase-separated system is centrifuged to accelerate migration of theanalyte.
 9. The method of claim 1, wherein the analyte migrates undergravitational forces.
 10. The method of claim 1, wherein the analytemigrates under buoyancy forces.
 11. The method of claim 1, wherein thephase-separated system is provided as dispersion or emulsion in anothercarrier phase.
 12. The method of claim 1, wherein the analyte ofinterest has a size of more than 200 nm.
 13. The method of claim 1,wherein after analyte migration the phases and the analyte are inthermodynamic equilibrium.
 14. The method of claim 1, wherein the phaseseparated system comprises three or more phases.
 15. The method of claim1, wherein the two or more phases comprise a common solvent which is anaqueous solvent.
 16. The method of claim 1, wherein the two or morephases comprise a common solvent which is an organic solvent.
 17. Themethod of claim 1, wherein the two or more phases comprise a commonsolvent which is a non-aqueous solvent selected from the groupsconsisting of liquid polymer, non-polar organic solvent, polar aproticor protic solvent, supercritical fluid, fuel, oils, and fluorinatedsolvents, and combinations thereof.
 18. The method of claim 17, whereinthe common solvent comprises dichloromethane.
 19. The method of claim 1,wherein the phase separated system comprises three or more phasesincluding the two or more phases comprising a common solvent which iswater and additional phase separated phases selected from the groupconsisting of organic solutions.
 20. The method of claim 15, wherein theaqueous solvent is selected from the group consisting of water, seawater, isotopes of water, buffered water, irrigation water, mineeffluent, colloidal solutions, emulsions, and a combination thereof. 21.The method of claim 1, wherein the analyte is selected from the groupconsisting of solid particles, an aggregate of particles, a liquid orgel immiscible in the solvent, a liquid crystal, and crystallinematerials.
 22. The method of claim 1, wherein the analyte is selectedfrom the group consisting of gem, bead, metal, glass, rock, mineral,crystal, plastic, bone, rubber, paper, fabric, coal, polymer particles,and gases.
 23. The method of claim 1, wherein the polymer is selectedfrom the group consisting of homopolymers, random copolymers, blockcopolymers, graft copolymers, ter-polymers, dendrimers, star polymersand combinations thereof.
 24. The method of claim 23, wherein thepolymer is linear, branched and/or cross-linked.
 25. The method of claim1, wherein the polymer is selected from the group consisting of dextran,dextran sulfate, chondroitin sulfate A, polysucrose,diethylaminoethyl-dextran, poly(2-vinylpyridine-N-oxide), polysucrose,poly(vinyl alcohol), poly(2-ethyl-2-oxazoline), poly(methacrylic acid),poly(ethylene glycol), polyacrylamide, polyethyleneimine, hydroxyethylcellulose, polyvinylpyrrolidone, carboxy-polyacrylamide, poly(acrylicacid), poly(2-acrylamido-2-methyl-1-propanesulfonic acid),poly(diallyldimethyl ammonium chloride), poly(styrene sulfonic acid),polyallylamine, alginic acid, poly(bisphenol A carbonate),polydimethylsiloxane, polystyrene, poly(4-vinylpyridine),polycaprolactone, polysulfone, poly(methyl methacrylate-co-methacrylicacid), poly(methyl methacrylate), poly(tetrahydrofuran), poly(propyleneglycol), and poly(vinyl acetate) and copolymers or terpolymers thereof.26. The method of claim 1, wherein the surfactant is selected from thegroup consisting of polysorbate,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate,polyoxyethylene-polyoxypropylene, 1-O-octyl-β-D-glucopyranoside,nonylphenol polyoxyethylene,4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,2-(Perfluoroalkyl)ethyl methacrylate, N,N-dimethyldodecylamine N-oxide,polyethylene glycol dodecyl ether, sodium dodecyl sulfate, sodiumcholate, benzylalkonium chloride and dodecyltrimethylammonium chloride.27. The method of claim 1, wherein one or more phases further comprisean additive selected from the group consisting of a co-solvent, an acid,a base, a miscible polymer, vitamin, drug, antibiotic, small molecule,dye, and fluorophore.
 28. The method of claim 1, wherein the sample isselected from the group consisting of a forensics study sample, a sampleindicative of animal health, a sample indicative of human identity usedfor border control, homeland security, or intelligence, a sample fromfood processing, a sample indicative of product quality, a sampleindicative of environmental safety, a sample containing differentcrystal polymorphs, and a combination thereof.
 29. The method of claim1, further comprising collecting the analyte from the boundary location.